Method of Expression Cloning in a Host Cell

Abstract

The invention relates to a promoter DNA sequence highly suited in an improved expression cloning method for isolation of DNA sequences comprising a DNA sequence encoding a protein of interest in a host cell and to the improved expression cloning method wherein use is made of this promoter. The isolated DNA sequences are useful in processes for producing a protein of interest.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a promoter highly suited for being used in a method for the identification of DNA sequences encoding proteins of interest by expression cloning.

BACKGROUND OF THE INVENTION

[0002] An increasing number of screening methods based on expression cloning are already known. Such methods have previously successfully been used for identification of prokaryotic gene products in e.g. Bacillus (cf. U.S. Pat. No. 4,469,791, WO2005/38024) and E. coli (e.g. WO 95/18219 and WO 95/34662). WO2005/38024 describes a method of screening protein secreting recombinant Bacillus cells. However, this method is lacking high throughput, automated detection technology. Yeast has also been used as a host for expression cloning of eukaryotic genes. Strasser et al. (Eur. J. Biochem. (1989)184: 699-706) have reported the identification of a fungal .alpha.-amylase by expression cloning of fungal genomic DNA in the yeast Saccharomyces cerevisiae. Similarly, WO 93/11249 reports the identification of a fungal cellulase by expression cloning of fungal cDNAs in S. cerevisiae. Lastly, an expression cloning method in filamentous fungus has been described (WO 99/32617).

[0003] In all these methods, large number of transformants would have to be screened before a transformant secreting a protein with properties of interest could be isolated. There is thus a need for an expression cloning method that would optimise the chance of detecting DNA sequences encoding secreted proteins.

BRIEF DESCRIPTION OF THE FIGURES

[0004] FIG. 1. Depicted is pPhtrA-gfp-amyE. This secretion reporter vector comprises the htrA promoter of B. subtilis in operative association with GFP. This vector is representative for pPhtrB-gfp-amyE.

[0005] FIG. 2. Construction of B. subtilis reporter strains for secretion-stress. The htr promoters were fused to the GFP gene, finally resulting in plasmids pPhtrA-gfp-amyE and pPhtrB-gfp-amyE. The resulting plasmids were integrated into the chromosome of Bacillus host cells via single crossing-over recombination in the corresponding htr loci, resulting in VT210A and VT210B cells. Cmr represents the chloramphenicol resistance gene.

[0006] FIG. 3. Depicted is pUBnpr-2, comprising the neutral protease gene npr (Genbank accession number K 02497) from Bacillus amyloliquefaciens.

[0007] FIG. 4. Depicted is pUBBAG, comprising the .beta.-glucanase gene bag (Genbank accession number M15674) from Bacillus amyloliquefaciens.

[0008] FIG. 5. Depicted is the analysis of Bacillus subtilis VT210A cells transformed with either plasmid pUB110 (empty control vector) or plasmid pKTH10 (encoding Bacillus amyloliquefaciens a-amylase gene amyQ) using Fluorescence Activated Cell Sorting. On the y-axis the amount of observed events is depicted, on the x-axis the relative fluorescence is depicted. In panel A, analysis of Bacillus subtilis VT210A cells transformed with empty vector pUB110 is depicted. In panel B, analysis of Bacillus subtilis VT210A cells transformed with amyQ vector pKTH10 is depicted. In panel C, analysis of a mixed population of Bacillus subtilis VT210A cells transformed with a ratio of pUB110 and pKTH10 of 1:20 is depicted. In panel D, cell sorting of a mixed population of Bacillus subtilis VT210A cells transformed with both pUB110 and pKTH10 is depicted; the cell sorting limit is indicated by a dashed vertical line. Cells demonstrating higher fluorescence than this limit were sorted.

[0009] FIG. 6. Depicted is the analysis of a mixed population of Bacillus subtilis VT210B cells transformed with either plasmid pUB110 (empty control vector) or plasmid pKTH10 (encoding Bacillus amyloliquefaciens a-amylase gene amyQ). Analysis was performed by Fluorescence Activated Cell Scanning. On the y-axis the amount of observed events is depicted, on the x-axis the relative fluorescence is depicted.

[0010] FIG. 7. Depicted is the pGBFINGFP-2 vector used for expression of Green Fluorescent Protein (GFP, Chalfie, M et al., Science (1994) 263(5148): 802-805) in A. niger. The backbone features of the depicted FIN vectors were previously described in WO 99/32617.

[0011] FIG. 8. Depicted is the pGBFIN-32 vector used for expression of phytase (PHY) (phytase gene PHY identical to FytA described in WO99/32617) in A. niger.

[0012] FIG. 9. Depicted is the pGBFIN-40 vector with the larger part of the phytase gene and the entire glucoamylase promoter removed, this vector is used as empty control vector for construction of A. niger WT-vector strain.

[0013] FIG. 10. Depicted is a Northern Blot analysis performed to confirm the identification of secretion-induced promoters.

[0014] FIG. 11. Depicted is a vector used for gene expression in A. niger. This vector is used as backbone in the construction of the secretion-induced A. niger reporter constructs.

[0015] FIG. 12. Depicted is the pGBFINGFPBLE-1 cloning vector. This secretion-inducible reporter construct comprises the GFP-BLE reporter fusion gene in operative association with secretion-inducible promoter 1 of the present invention. The construct is representative for pGBFINGFPBLE-2, pGBFINGFPBLE-3, pGBFINGFPBLE-4, and pGBFINGFPBLE-5.

[0016] FIG. 13. Depicted is pGBTOPSEL-1, this vector is used as the backbone for construction of the final secretion-inducible reporter plasmids. Backbone features of the depicted vector were previously described in WO 99/32617.

[0017] FIG. 14. Depicted is the final secretion reporter construct pGBTOPGFPBLE-1. This secretion-induced reporter vector comprises the GFP-BLE reporter construct in operative association with secretion-inducible promoter 1 of the present invention. The vector is representative for pGBTOPGFPBLE-2, pGBTOPGFPBLE-3, pGBTOPGFPBLE-4, and pGBTOPGFPBLE-5.

[0018] FIG. 15. Depicted is the difference in phleomycin resistance of A. niger co-transformants containing a reporter construct with secretion-inducible promoter P4 in combination with a library construct encoding either an intracellular protein (represented by black dots) or an extracellular protein (represented by black squares).

[0019] FIG. 16. Depicted is the difference in phleomycin resistance of A. niger co-transformants containing a reporter construct with secretion-inducible promoter P5 in combination with a library construct encoding either an intracellular protein (represented by black dots) or an extracellular protein (represented by black squares).

[0020] FIG. 17. Depicted is an E-PAGE.TM. gel, wherein each lane represents a co-transformed A. niger clone comprising a reporter contruct with secretion-inducible promoter P1 in combination with a library construct encoding an extracellular protein. M represents the E-PAGE SeeBlue.RTM. prestained standard (Invitrogen, U.K.).

[0021] FIG. 18. Depicted is an E-PAGE.TM. gel, wherein each lane represents a co-transformed A. niger clone comprising a reporter contruct with secretion-inducible promoter P3 in combination with a library construct encoding an extracellular protein. M represents the E-PAGE SeeBlue.RTM. prestained standard (Invitrogen, U.K.).

DESCRIPTION OF THE INVENTION

Promoter DNA Sequence

[0022] According to a first aspect of the invention, there is provided a promoter DNA sequence such as: [0023] (a) a DNA sequence as presented in the following list: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, [0024] (b) a DNA sequence capable of hybridizing with a DNA sequence of (a), [0025] (c) a DNA sequence being at least 50% homologous to a DNA sequence of (a), [0026] (d) a variant of any of the DNA sequences of (a) to (c), or [0027] (e) a subsequence of any of the DNA sequences of (a) to (d).

[0028] According to a preferred embodiment, the promoter sequence of the invention is one promoter DNA sequence such as: [0029] (a) one DNA sequence as presented in the following list: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, [0030] (b) one DNA sequence capable of hybridizing with a DNA sequence of (a), [0031] (c) one DNA sequence being at least 50% homologous to a DNA sequence of (a), [0032] (d) one variant of any of the DNA sequences of (a) to (c), or [0033] (e) one subsequence of any of the DNA sequences of (a) to (d).

[0034] In the context of this application, a promoter DNA sequence is a DNA sequence, which is capable of controlling the expression of a coding sequence, when this promoter DNA sequence is in operative association with this coding sequence. The term "in operative association" is defined herein as a configuration in which a promoter DNA sequence is appropriately placed at a position relative to a coding sequence such that the promoter DNA sequence directs the production of a polypeptide encoded by the coding sequence.

[0035] The term "coding sequence" is defined herein as a nucleic acid sequence that is transcribed into mRNA, which is translated into a polypeptide when placed under the control of the appropriate control sequences. The boundaries of the coding sequence are generally determined by the ATG start codon, which is normally the start of the open reading frame at the 5' end of the mRNA and a transcription terminator sequence located just downstream of the open reading frame at the 3' end of the mRNA. A coding sequence can include, but is not limited to, genomic DNA, cDNA, semisynthetic, synthetic, and recombinant nucleic acid sequences. Preferably, a promoter DNA sequence is defined by being the DNA sequence located upstream of a coding sequence associated thereto and by being capable of controlling the expression of this coding sequence.

[0036] More specifically, the term "promoter" is defined herein as a DNA sequence that binds the RNA polymerase and directs the polymerase to the correct downstream transcriptional start site of a coding sequence encoding a polypeptide to initiate transcription. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region. The term "promoter" will also be understood to include the 5' non-coding region (between promoter and translation start) for translation after transcription into mRNA, cis-acting transcription control elements such as enhancers, and other nucleotide sequences capable of interacting with transcription factors.

[0037] In a preferred embodiment, the promoter DNA sequence of the invention is a DNA sequence derived from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.

[0038] According to another preferred embodiment, the promoter DNA sequence of the invention is a DNA sequence capable of hybridizing with a DNA sequence as presented in the following list: SEQ ID NO 1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 and which still retains promoter activity.

[0039] Promoter activity is preferably determined by measuring the concentration of the protein(s) produced as a result of the expression of a coding sequence(s), which is (are) in operative association with the promoter. Alternatively the promoter activity is determined by measuring the enzymatic activity of the protein(s) coded by the coding sequence(s), which is (are) in operative association with the promoter. According to a preferred embodiment, the promoter activity (and its strength) is determined by measuring the expression of the coding sequence of the lacZ reporter gene (In Luo, Gene 163 (1995) 127-131 or Perkins and Youngman (1986) Proc Natl Acad Sci USA 83: 140; or Vagner et al. (1998) Microbiology 144: 3097). According to another preferred embodiment, the promoter activity is determined by using the green fluorescent protein as coding sequence (In Microbiology. 1999 March; 145 (Pt 3):729-34. Santerre Henriksen A L, Even S, Muller C, Punt P J, van den Hondel C A, Nielsen J.Study). In bacterial cells, GFP and other fluorescent proteins have been used extensively for expression and (dynamic) protein localization studies (reviewed in Southward and Surette (2002) Mol. Microbiol. 45: 1191). Additionally, promoter activity can be determined by measuring the mRNA levels of the transcript generated under control of the promoter. The mRNA levels can, for example, be measured by Northern blot, GeneChips.TM. (Affymetrix) or spotted array technology or quantitative (real time) PCR (J. Sambrook, E. F. Fritsch, and T. Maniatis, 2001, Molecular Cloning, A Laboratory Manual, 3d edition, Cold Spring Harbor, N.Y.). Most preferably, promoter activity is determined by mRNA analysis using Northern blot.

[0040] Preferably, (isolated) promoter DNA sequences of the invention hybridize under very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with a nucleic acid probe which hybridizes under the same conditions with:

(i) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, or

[0041] (ii) a subsequence of (i), or (iii) a complementary strand of (i), (ii),

[0042] The term complementary strand is known to the person skilled in the art and is described in (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 may be ranged between 20 nucleotides and the respective whole sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 upstream the coding region (the corresponding coding regions of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 are respectively given in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29).

[0043] The nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 or a subsequence thereof (as defined in the previous paragraph) may be used to design a nucleic acid probe to identify and clone DNA promoters from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, preferably at least 25, and more preferably at least 35 nucleotides in length. Additionally, such probes can be used to amplify DNA promoters though PCR. Longer probes can also be used. DNA, RNA and Peptide Nucleid Acid (PNA) probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with @32 P, @33 P @3H, @35 S, biotin, or avidin or a fluorescent marker). Such probes are encompassed by the present invention.

[0044] Thus, a genomic DNA or cDNA library prepared from such other organisms may be screened for DNA, which hybridizes with the probes described above and which encodes a polypeptide. Genomic or other DNA from such other organisms may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA sequence which is homologous with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 or a subsequence thereof, the carrier material may be used in a Southern blot. For purposes of the present invention, hybridization indicates that the DNA sequence hybridizes under very low to very high stringency conditions to a labeled nucleic acid probe corresponding to the DNA sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, to the complementary strands of said DNA sequences, or to subsequences of said DNA sequences. Molecules to which the nucleic acid probe hybridizes under these conditions are detected using for example a X-ray film. Other hybridisation techniques also can be used, such as techniques using fluorescence for detection and glass sides and/or DNA microarrays as support. An example of DNA microarray hybridisation detection is given in FEMS Yeast Res. 2003 December; 4(3):259-69 (Daran-Lapujade P, Daran J M, Kotter P, Petit T, Piper M D, Pronk J T. "Comparative genotyping of the Saccharomyces cerevisiae laboratory strains S288C and CEN.PK113-7D using oligonucleotide microarrays". Additionally, the use of PNA microarrays for hybridization is described in Nucleic Acids Res. 2003 October 1; 31(19):e119 (Brandt O, Feldner J, Stephan A, Schroder M, Schnolzer M, Arlinghaus H F, Hoheisel J D, Jacob A. PNA microarrays for hybridisation of unlabelled DNA samples.)

[0045] In a preferred embodiment, the nucleic acid or DNA probe is the DNA sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. In another embodiment, the nucleic acid probe is the DNA sequence having nucleotides 100 to 150 of SEQ ID NO:1 or the DNA sequence having nucleotides 170 to 220 of SEQ ID NO:2.

[0046] In another preferred embodiment, the nucleic acid probe is the DNA sequence having: [0047] nucleotides 996 to 2797 of SEQ ID NO: 20, nucleotides 996 to 3561 of SEQ ID NO: 21, nucleotides 997 to 3536 of SEQ ID NO: 22, nucleotides 1034 to 3175 of SEQ ID NO: 23, or nucleotides 1080 to 4054 of SEQ ID NO: 24, more preferably: [0048] nucleotides 1496 to 2497 of SEQ ID NO: 20, nucleotides 1496 to 3261 of SEQ ID NO: 21, nucleotides 1497 to 3236 of SEQ ID NO: 22, nucleotides 1534 to 2875 of SEQ ID NO: 23, or nucleotides 1580 to 3754 of SEQ ID NO: 24, even more preferably: [0049] nucleotides 1896 to 2197 of SEQ ID NO: 20, nucleotides 1896 to 2861 of SEQ ID NO: 21, nucleotides 1897 to 2936 of SEQ ID NO: 22, nucleotides 1934 to 2575 of SEQ ID NO: 23, or nucleotides 1980 to 3454 of SEQ ID NO: 24, and most preferably: [0050] nucleotides 1996 to 2100 of SEQ ID NO: 20, nucleotides 1996 to 2300 of SEQ ID NO: 21, nucleotides 1997 to 2400 of SEQ ID NO: 22, nucleotides 2034 to 2100 of SEQ ID NO: 23, or nucleotides 2080 to 2800 of SEQ ID NO: 24.

[0051] According to another preferred embodiment, the probe is part of the DNA sequence of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 located upstream of the coding sequence. The corresponding coding regions of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 are respectively given in SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29).

[0052] For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42 degrees Celsius in 5 times SSPE, 0.3% SDS, 200 microgram/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures. For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2 times SSC, 0.2% SDS preferably at least at 45 DEG C. (very low stringency), more preferably at least at 50 degrees Celsius (low stringency), more preferably at least at 55 degrees Celsius (medium stringency), more preferably at least at 60 degrees Celsius (medium-high stringency), even more preferably at least at 65 degrees Celsius (high stringency), and most preferably at least at 70 degrees Celsius (very high stringency).

[0053] For short probes which are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at 5 degrees Celsius to 10 degrees Celsius below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures. For short probes, which are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6 times SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6 times SSC at 5 degrees Celsius to 10 degrees Celsius below the calculated Tm.

[0054] According to another preferred embodiment, the promoter DNA sequence of the invention derived from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 is first used to clone the native gene, coding sequence or part of it, which is operatively associated with it. This can be done starting with either SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 or a subsequence thereof as earlier defined and using this sequence as a probe. The probe is hybridised to a cDNA or a genomic library of a given host either Bacillus, Aspergillus niger or any other host as defined in this application. Once the native gene or part of it has been cloned, it can be subsequently used itself as a probe to clone homologous genes thereof derived from other host by hybridisation experiments as described herein. In this context, a homologous gene means a gene, which is at least 50% homologous to the native gene. Preferably, the homologous gene is at least 55% homologous, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, even more preferably at least 75% preferably about 80%, more preferably about 90%, even more preferably about 95%, and most preferably about 97% homologous to the native gene.

[0055] The sequence upstream the coding sequence of the homologous gene is a promoter encompassed by the present invention. Alternatively, the sequence of the native gene, coding sequence or part of it, which is operatively associated with a promoter of the invention can be identified by using SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29 or a subsequence thereof as earlier defined to search genomic databases using for example an alignment or BLAST algorithm as described herein. This identified sequence subsequently can be used to identify orthologues or homologous genes in any other fungal host as defined in this application. The sequence upstream the coding sequence of the identified orthologue or homologous gene is a promoter encompassed by the present invention.

[0056] According to another preferred embodiment, the promoter DNA sequence of the invention is a(n) (isolated) DNA sequence derived from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 and which still has promoter activity as defined earlier.

[0057] According to another preferred embodiment, the promoter DNA sequence of the invention is a(n) (isolated) DNA sequence derived from either SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24, respectively, which is at least 50% homologous to at least part of the respective corresponding DNA sequences: SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19 situated upstream of the respective coding regions (SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29). Preferably, the derived DNA sequence is at least 55% homologous, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, even more preferably at least 75% preferably about 80%, more preferably about 90%, even more preferably about 95%, and most preferably about 97% homologous to at least part of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19 situated upstream of the respective corresponding coding region.

[0058] For purposes of the present invention, the degree of homology between two nucleic acid sequences is preferably determined by the Wilbur-Lipman method (Wilbur and Lipman, 1983, Proceedings of the National Academy of Science USA 80: 726-730) using the LASERGENE.TM. MEGALIGN.TM. software (DNASTAR, Inc., Madison, Wis.) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10. Pairwise alignment parameters were Ktuple=3, gap penalty=3, and windows=20.

[0059] According to another preferred embodiment, the promoter DNA sequence of the invention is derived from a(n) (isolated) DNA sequence, which is a variant of either SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19 or part of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19 situated upstream of the respective corresponding coding region. The term "variant promoter" is defined herein as a promoter having a nucleotide sequence comprising a substitution, deletion, and/or insertion of one or more nucleotides of a parent promoter, wherein the variant promoter has more or less promoter activity than the corresponding parent promoter. The term "variant promoter" will also encompass natural variants and in vitro generated variants obtained using methods well known in the art such as classical mutagenesis, site-directed mutagenesis, and DNA shuffling. A variant promoter may have one or more mutations. Each mutation is an independent substitution, deletion, and/or insertion of a nucleotide. The introduction of a substitution, deletion, and/or insertion of a nucleotide into the promoter may be accomplished using any of the methods known in the art such as classical mutagenesis, site-directed mutagenesis, or DNA shuffling. Particularly useful is a procedure, which utilizes a supercoiled, double stranded DNA vector with an insert of interest and two synthetic primers containing the desired mutation. The oligonucleotide primers, each complementary to opposite strands of the vector, extend during temperature cycling by means of Pfu DNA polymerase. On incorporation of the primers, a mutated plasmid containing staggered nicks is generated. Following temperature cycling, the product is treated with Dpnl, which is specific for methylated and hemimethylated DNA to digest the parental DNA template and to select for mutation-containing synthesized DNA. Other procedures known in the art may also be used. Example of other procedures are the QuickChange.TM. site-directed mutagenesis kit (Stratagene Cloning Systems, La Jolla, Calif.), the `The Altered Sites.RTM. II in vitro Mutagenesis Systems` (Promega Corporation) or by overlap extension using PCR as described in Gene. 1989 Apr. 15; 77(1):51-9. (Ho S N, Hunt H D, Horton R M, Pullen J K, Pease L R "Site-directed mutagenesis by overlap extension using the polymerase chain reaction") or using PCR as described in Molecular Biology: Current Innovations and Future Trends. (Eds. A. M. Griffin and H. G. Griffin. ISBN 1-898486-01-8 1995 Horizon Scientific Press, PO Box 1, Wymondham, Norfolk, U.K.). According to a preferred embodiment, the variant promoter is a promoter, which has at least one modified regulatory site as compared to the promoter sequence first derived from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19. Such regulatory site can have been removed in its entirety or specifically mutated as explained above. The regulation of such promoter variant is thus modified so that for example it is no longer induced by glucose. Examples of such promoter variants and techniques on how to obtain them are described in EP 673 429B or in WO 94/04673.

[0060] The promoter variant can be an allelic variant. An allelic variant denotes any of two or more alternative forms of a gene occupying the same chomosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. The variant promoter may be obtained by the following method comprising steps (a) and (b):

(a) hybridizing a DNA sequence under very low, low, medium, medium-high, high, or very high stringency conditions with: [0061] (i) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, or [0062] (ii) a subsequence of (i), or [0063] (iii) a complementary strand of (i), (ii), and (b) isolating the variant promoter from the DNA sequence. Stringency and wash conditions are defined herein.

[0064] The variant promoter can be a promoter, whose sequence may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the promoter sequence with the coding region of the nucleic acid sequence encoding a polypeptide.

[0065] In another preferred embodiment, the promoter DNA sequence is derived from a subsequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19. A subsequence is preferably defined in this context as being part of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19 located upstream of the respective corresponding coding sequence as defined previously.

[0066] The subsequence preferably contains at least about 50 nucleotides, or preferably at least about 100 nucleotides, more preferably at least about 200 nucleotides, even more preferably at least about 230 nucleotides, even more preferably at least about 250 nucleotides, and most preferably at least about 290 nucleotides.

[0067] According to another preferred embodiment, the subsequence differs from the one defined in former sentence by the fact that one or more nucleotides from the 5' and/or 3' end have been deleted, said DNA sequence still having promoter activity as defined earlier.

[0068] In another preferred embodiment, the promoter subsequence is a `trimmed` subsequence from translation start and/or from transcription start. An example of trimming a promoter and functionally analysing it is described in Gene. 1994 Aug. 5; 145(2):179-87: the effect of multiple copies of the upstream region on expression of the Aspergillus niger glucoamylase-encoding gene. Verdoes J C, Punt P J, Stouthamer A H, van den Hondel C A).

[0069] The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The specific sequences disclosed herein can be readily used and be eventually subjected to further sequence analyses thereby identifying sequencing errors to isolate the original DNA sequence from a filamentous fungus, in particular Aspergillus niger.

[0070] Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.

[0071] The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.

[0072] Functional nucleic acid equivalents may typically contain mutations that do not alter the biological function of the promoter it contains. The term "functional equivalents" also encompasses orthologues of the A. niger DNA sequences. Orthologues of the A. niger DNA sequences are DNA sequences that can be isolated from other strains or species and possess a similar or identical biological activity.

[0073] Homologous (similar or identical) sequences can also be determined by using a "sequence comparison algorithm". Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l Acad. Sci. USA 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. An example of an algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul, et al., J. Mol. Biol. 215: 403-410 (1990). Alternatively other programs can be used for sequence alignment as earlier described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. These initial neighborhood word hits act as starting points to find longer HSPs containing them. The word hits are expanded in both directions along each of the two sequences being compared for as far as the cumulative alignment score can be increased. Extension of the word hits is stopped when: the cumulative alignment score falls off by the quantity X from a maximum achieved value; the cumulative score goes to zero or below; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm then performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90: 5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, an amino acid sequence is considered similar to a protein such as a protease if the smallest sum probability in a comparison of the test amino acid sequence to a protein such as a protease amino acid sequence is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0074] Preferably the similarity of the variant promoter is at least 40% homology to one of the DNA sequences having SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19. More preferably the similarity is at least 50%, more preferably, at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90% and more preferably at least 95% or at least 98% homology to the DNA sequence having SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19.

[0075] In addition to naturally occurring allelic variants of the promoter sequence, the skilled person will recognise that changes can be introduced by mutation into the promoter sequence derived from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 or SEQ ID NO:19, without substantially altering its promoter function.

[0076] The promoter sequences of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide is produced by the source or by a cell in which a gene from the source has been inserted. Preferably, the microorganism is a prokaryote. Preferred prokaryotes are Bacillus and E. coli. Preferred Bacilli are Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus alcalophilus, Bacillus clausii, Bacillus brevis, Bacillus circulans, Bacillus firmus, Bacilluspumilis, Bacillus stearothermophilus, Bacillus megaterium, Bacillus lentus, or Bacillus thuringiensis.

[0077] Alternatively, the promoter sequence is obtained from an eukaryote, preferably a fungal source, and more preferably from a yeast strain such as a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia strain; or more preferably from a filamentous fungal strain such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma strain.

[0078] In a preferred embodiment, the promoter sequences are obtained from a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Saccharomyces bayanus, Saccharomyces bayanus, var. uvarum or Saccharomyces pastorianus strain.

[0079] In another preferred embodiment, the promoter sequences are obtained from an Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, A. nidulans, A. niger, preferably A. niger CBS 513.88, A. sojae, Aspergillus oryzae (A. oryzae), Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride strain.

[0080] In another preferred embodiment, the promoter sequences are obtained from a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum strain.

[0081] It will be understood that for the aforementioned species, the invention encompasses the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents. Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0082] Furthermore, such nucleic acid sequences may be identified and obtained from other sources including microorganisms isolated from nature (e.g, soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The nucleic acid sequence may then be derived by similarly screening a genomic DNA library of another microorganism. Once a nucleic acid sequence encoding a promoter has been detected with the probe(s), the sequence may be isolated or cloned by utilizing techniques which are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

[0083] In the present invention, the promoter DNA sequence may also be a hybrid promoter comprising a portion of one or more promoters of the present invention; a portion of a promoter of the present invention and a portion of another known promoter, e.g., a leader sequence of one promoter and the transcription start site from the other promoter; or a portion of one or more promoters of the present invention and a portion of one or more other promoters. The other promoter may be any promoter sequence, which shows transcriptional activity in the host cell of choice including a variant, truncated, and hybrid promoter, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The other promoter sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide and native or foreign to the cell.

[0084] As a preferred embodiment, important regulatory subsequences of the promoter identified can be fused to other `basic` promoters to enhance their promoter activity (as for example described in Mol. Microbiol. 1994 May; 12(3):479-90. Regulation of the xylanase-encoding xInA gene of Aspergillus tubigensis. de Graaff L H, van den Broeck H C, van Ooijen A J, Visser J.).

[0085] Other examples of other promoters useful in the construction of hybrid promoters with the promoters of the present invention include the promoters obtained from the genes for A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A. niger or Aspergillus awamori glucoamylase (glaA), A. niger gpdA, A. niger glucose oxidase goxc, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (WO 96/00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for A. niger neutral alpha-amylase and A. oryzae triose phosphate isomerase), Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase, and mutant, truncated, and hybrid promoters thereof. Other useful promoters for yeast host cells are described by Romanoset al., 1992, Yeast 8: 423-488. Other useful promoters for bacterial cells are the promoter of the following genes B. subtilis alkaline protease (apr, B. subtilis neutral protease (npr, B. amyloliquefaciens .alpha.-amylase (amyQ), B. amyloliquefaciens alkaline protease (apr, and B. amyloliquefaciens neutral protease (npr)

[0086] In the present invention, the promoter DNA sequence may also be a "tandem promoter". A "tandem promoter" is defined herein as two or more promoter sequences each of which is in operative association with a coding sequence and mediates the transcription of the coding sequence into mRNA.

[0087] The tandem promoter comprises two or more promoters of the present invention or alternatively one or more promoters of the present invention and one or more other known promoters, such as those exemplified above useful for the construction of hybrid promoters. The two or more promoter sequences of the tandem promoter may simultaneously promote the transcription of the nucleic acid sequence. Alternatively, one or more of the promoter sequences of the tandem promoter may promote the transcription of the nucleic acid sequence at different stages of growth of the cell or, in the case of fungal hosts, morphological different parts of the mycelia, or different cellular compartments during spore development in sporulating bacteria such as bacilli.

[0088] In the present invention, the promoter may be foreign to the coding sequence and/or to the host cell. A variant, hybrid, or tandem promoter of the present invention will be understood to be foreign to a DNA sequence encoding a polypeptide even if the wild-type promoter is native to the coding sequence or to the host cell.

[0089] A variant, hybrid, or tandem promoter of the present invention has at least about 20%, preferably at least about 40%, more preferably at least about 60%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 100%, even more preferably at least about 200%, most preferably at least about 300%, and even most preferably at least about 400% of the promoter activity of any promoter derived from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24 and defined earlier. The promoter activity is preferably determined as defined earlier. Most preferably, promoter activity is determined by mRNA analysis using Northern blot.

DNA Construct

[0090] According to a second aspect, the invention provides a DNA construct comprising a promoter DNA sequence of the invention as defined in the former section operatively associated with a reporter gene conferring a selectable trait. The reporter gene can be any gene conferring a selectable trait to a suitable host. Preferably, the reporter gene is a selection marker gene.

[0091] "DNA construct" is defined herein as a nucleic acid molecule, either single or double-stranded, which is isolated from a naturally occurring gene or which has been modified to contain segments of nucleic acid combined and juxtaposed in a manner that would not otherwise exist in nature.

[0092] The term DNA construct is synonymous with the term expression cassette when the DNA construct contains a coding sequence or reporter gene and all the control sequences required for expression of the coding sequence or reporter gene. In the context of this application the term "DNA contruct" is interchangeably used with the term "isolated DNA construct" wherein both terms are contrued to be synonymous.

[0093] The DNA construct may comprise a selection marker gene, which permits easy selection of transformed host cells. In case the DNA construct comprises two selection marker genes, the skilled person will understand that these two selection marker genes are not identical: the first one associated with the promoter of the invention is used in the screening method of the invention and the second one present on the DNA construct allows a standard selection of transformed host cells.

[0094] The reporter gene is in operative association with the promoter DNA sequence of the invention such that the reporter gene can be expressed under the control of the promoter DNA sequence in a given host cell. The polypeptide encoded by the reporter gene may be native or heterologous to the host cell and/or to the promoter DNA sequence.

[0095] In this context "a" means "at least one". Therefore, the DNA construct comprises at least one reporter gene. The host may be co-transformed with at least two DNA constructs, one comprising the selection marker and another one comprising the promoter of the invention in association with a reporter gene.

[0096] Alternatively, and according to a more preferred embodiment, the reporter gene comprised in the DNA construct is a hybrid reporter gene comprising at least part of a first reporter gene with at least part of a second reporter gene, wherein the coding sequences of the first and second reporter genes are in-frame coupled to each other and wherein said hybrid reporter gene is operably linked to the promoter of the invention. The first and/or second reporter gene may be any gene conferring a selectable trait to a suitable host. A preferred hybrid reporter gene for fungi comprises the GFP gene and the phleomycin resistance gene (BLE) of Streptoalloteichus hindustanis (Drocourt et al., NAR, 1990).

[0097] A selection marker or selectable marker is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Suitable selectable markers for prokaryotic host cells include, but are not limited to, kanamycin, neomycin, erythromycin and other MLS-type markers (macrolide- lincosamide-streptogramin B), chloramphenicol, ampicillin, tetracyclin, streptomycin and spectinomycin. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a flamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hygB (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), trpC (anthranilate synthase), as well as equivalents thereof. Marker conferring resistance against e.g. phleomycin, hygromycin B or G418 can also be used. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of A. nidulans or A. oryzae and the bar gene of Streptomyces hygroscopicus. The amdS marker gene is preferably used applying the technique described in EP 635 574B or WO 97/06261. A preferred selection marker gene is the A. nidulans amdS coding sequence fused to the A. nidulans gpdA promoter (EP635 574B). AmdS genes from other filamentous fungus may also be used (WO 97/06261).

[0098] According to another preferred embodiment, the reporter gene encodes a selectable marker for prokaryotic, yeast and/or filamentous fungal cells as described here above. Alternatively, the reporter gene encodes a reporter protein like: beta-galactosidase, beta-glucuronidase, aequorin, Green Fluorescent Protein (GFP) and variants thereof (Red, Cyan, Yellow-fluorescent protein), luciferase, lux, heme, beta-lactamase, or alkaline phosphatase. More preferably, the reporter gene is or comprises a gene encoding a fluorescent reporter protein such as, but not limited to GFP, YFP, and CFP (green, yellow and cyan fluorescent proteins, respectively). Transformants will exhibit fluorescence and can be separated from non expressing clones using sensitive cell sorting technology (FACS, fluorescence activated cell sorting). An important advantage of this method is that plating of transformation mixtures is not required (which considerably increases the throughput of the screening procedure) and that positive clones can arrayed directly into an MTP format for further automated processing. Alternatively, the reporter gene encodes a (trans)membrane protein or a cell wall protein to which antibodies can be raised. Transformants exhibiting said membrane protein can be separated from non expressing transformants using magnetic cell sorting technologies (MACS.RTM., Magnetic cell sorting, www.miltenyibiotec.com).

Control Elements

[0099] The DNA construct may further comprise one or more control sequences in addition to the promoter DNA sequence, which direct the expression of the reporter gene in a suitable host cell under conditions compatible with the control sequences. Expression will be understood to include any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. One or more control sequences may be native to the reporter gene or to the host. Alternatively, one or more control sequences may be replaced with one or more control sequences foreign to the nucleic acid sequence for improving expression of the reporter gene in a host cell.

[0100] The term "control sequences" is defined herein to include all components, including the promoter of the invention, which are necessary or advantageous for the expression of a coding sequence such as a reporter gene. Each control sequence may be native or foreign to the coding sequence encoding a polypeptide. Such control sequences include, but are not limited to, a leader, optimal translation initiation sequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870), polyadenylation sequence, propeptide sequence, signal peptide sequence, upstream activating sequence, the promoter of the invention including variants, fragments, hybrid and tandem thereof and transcription terminator. At a minimum, the control sequences include transcriptional and translational stop signals and (part of) the promoter of the invention. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding sequence, such as a reporter gene, encoding a polypeptide.

[0101] The control sequence may be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is in operative association with the 3' terminus of the coding sequence encoding the polypeptide. Any terminator, which is functional in the host cell of choice may be used in the present invention.

[0102] The skilled person would know that 5' end and 3' end of a sequence is defined for each coding sequence. For a given terminator sequence and a given coding sequence, the skilled person would know how to operatively associate them together.

[0103] Preferred terminators for filamentous fungal host cells are obtained from the genes for A. oryzae TAKA amylase, A. niger glucoamylase, A. nidulans anthranilate synthase, A. niger alpha-glucosidase, trpc gene, and Fusarium oxysporum trypsin-like protease.

[0104] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al, 1992, supra. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is in operative association with the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.

[0105] Preferred leaders for filamentous fungal host cells are obtained from the genes for A. oryzae TAKA amylase, A. nidulans triose phosphateisomerase and A. niger glucoamylase.

[0106] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase (ADH2 and GAP).

[0107] The control sequence may also be a polyadenylation sequence, a sequence in operative association with the 3' terminus of the nucleic acid sequence and which, when Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for A. oryzae TAKA amylase, A. niger glucoamylase, A. nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and A. niger alpha-glucosidase.

[0108] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

[0109] It may also be desirable to add regulatory sequences, which allow the regulation of the expression of the reporter gene relative to the growth of the host cell. Examples of regulatory systems are those which cause the expression of the reporter gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, and trp operator systems of E. coli, and the spac, xyl, sacB, and citM systems in B. subtilis (reviewed in Meima et al. (2004) Expression Systems in Bacillus. In "Protein Expression Technologies. Current status and future trends" (F. Baneyx, ed.), Horizon Bioscience, Wymondham, Norfolk, UK, pp. 199-252). Other promoters useful for controlled expression in bacilli are those induced under nutrient starvation conditions such as the Pho regulon and TnrA/GlnR system, which are under control of phosphate aid nitrogen, respectively. In yeast, the ADH2 system or GALL system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, A. niger glucoamylase promoter, A. oryzae glucoamylase promoter, A. tubingensis endoxylanase (xlnA) promoter, A. niger nitrate reductase (niaD) promoter, Trichoderma reesei cellobiohydrolase promoter and the A. nidulans alcohol and aldehyde dehydrogenase (alcA and aldA, respectively) promoters as described in U.S. Pat. No. 5,503,991) may be used as regulatory sequences. Other examples of regulatory sequences are those, which allow for gene amplification. In eukaryotic systems, these include the dihydrofolate reductase gene, which is amplified in the presence of methotrexate, and the metallothionein genes, which are amplified with heavy metals. In these cases, the nucleic acid sequence encoding the polypeptide would be in operative association with the regulatory sequence.

[0110] Endogenous regulatory sequence(s) present in the promoter DNA sequence of the invention may be removed, for example removal of creA binding sites (carbon catabolite repression as described earlier in EP673429B), change of pacC and areA (for pH and nitrogen regulation). In B. subtilis, carbon catabolite repression (CCR) involves binding of the CcpA protein to the cre element. The cre element was used in combination with the xylose inducible xyl promoter for the development of an expression system, allowing for dual control of genes of interest (Bhavsar et al. (2001) Appl Environ Microbiol 67: 403).

[0111] Preferably, the DNA construct comprises a promoter DNA sequence from the invention, a reporter gene in operative association with said promoter DNA sequence and translational control sequences such as: [0112] one translational termination sequence orientated in 5' towards 3' direction selected from the following list of sequences: TAAG, TAGA and TAAA, preferably TAM, and/or [0113] one translational initiator coding sequence orientated in 5' towards 3' direction selected from the following list of sequences: GCTACCCCC; GCTACCTCC; GCTACCCTC; GCTACCTTC; GCTCCCCCC; GCTCCCTCC; GCTCCCCTC; GCTCCCTTC; GCTGCCCCC; GCTGCCTCC; GCTGCCCTC; GCTGCCTTC; GCTTCCCCC; GCTTCCTCC; GCTTCCCTC; and GCTTCCTTC, preferably GCT TCC TTC, and/or [0114] one transcriptional initiator sequence selected from the following list of sequences: 5'-mwChkyCAAA-3'; 5'-mwChkyCACA-3' or 5'-mwChkyCMG-3', using ambiguity codes for nucleotides: m (A/C); w (A/T); y (C/T); k (G/T); h (A/C/T), preferably 5'-CACCGTCAAA-3' or 5'-CGCAGTCAAG-3'.

[0115] In the context of this invention, the term "translational initiator coding sequence" is defined as the nine nucleotides immediately downstream of the initiator or start codon of the open reading frame of a DNA coding sequence. The initiator or start codon encodes for the AA methionine. The initiator codon is typically ATG, but may also be any functional start codon such as GTG.

[0116] In the context of this invention, the term "translational termination sequence" is defined as the three or four nucleotides starting from the translational stop codon at the 3' end of the open reading frame or nucleotide coding sequence and oriented in 5' towards 3' direction.

[0117] In the context of this invention, the term "translational initiator sequence" is defined as the ten nucleotides immediately upstream of the initiator or start codon of the open reading frame of a DNA sequence coding for a polypeptide. The initiator or start codon encodes for the AA methionine. The initiator codon is typically ATG, but may also be any functional start codon such as GTG. It is well known in the art that uracil, U, replaces the deoxynucleotide thymine, T, in RNA.

[0118] Preferred control sequences for prokaryotes have already been described in EP 284 126A.

[0119] The present invention also relates to recombinant expression vectors comprising a promoter of the present invention operatively associated with a reporter gene, and transcriptional and translational stop signals. The various reporter gene and control sequences described above may be joined together to produce a recombinant expression vector which may include one or more convenient restriction sites to allow for insertion or substitution of the promoter and/or reporter gene at such sites. Alternatively, fusion of reporter gene and promoter can be done by e.g. sequence overlap extension using PCR (SOE-PCR), as described in Gene. 1989 Apr. 15; 77(1):51-9. Ho S N, Hunt H D, Horton R M, Pullen J K, Pease L R "Site-directed mutagenesis by overlap extension using the polymerase chain reaction") or by cloning using the Gateway.TM. cloning system (Invitrogen).

[0120] The recombinant expression vector may be any vector capable of transforming a host cell. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids. The vectors may be integrative or autonomously replicating vectors.

[0121] The vector may be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The autonomously replicating vector may contain any means for assuring self-replication. The vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used. An example of an autonomously maintained cloning vector is a cloning vector comprising the AMA1-sequence. AMAL is a 6.0-kb genomic DNA fragment isolated from A. nidulans, which is capable of Autonomous Maintenance in Aspergillus (see e.g. Aleksenko and Clutterbuck (1997), Fungal Genet. Biol. 21: 373-397). Examples of autonomously replicating vectors useful for expression in Gam-positive bacteria such as B. subtilis and B. amyloliquefaciens are the staphylococcal vector pUB110 (McKenzie et al. (1986) Plasmid 15: 93) and the endogenous B. subtilis plasmids pTA1015, pTA1040 and pTA1060 and vectors derived thereof (e.g. pHB201; Bron et al. (1998) J Biotechnol 64: 3).

[0122] The vectors of the present invention preferably contain (an) element(s) that permits stable integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0123] For integration into the host cell genome, the vector may rely on the promoter sequence and/or reporter gene sequence or any other element of the vector for stable integration of the vector into the genome by homologous or non-homologous recombination. The vector may contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 20 to 50, preferably, 55 to 95, preferably 100 to 1,500 base pairs, preferably 400 to 1,500 base pairs, more preferably 800 to 1,500 base pairs, and most preferably at least 2 kb, which are homologous to a DNA sequence in a predetermined target locus in the genome of the host cell. The integrational elements may be any sequence that is homologous with the target sequence in the genome d the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. In order to promote targeted integration, the cloning vector is preferably linearized prior to transformation of the host cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus.

[0124] Preferably, the integrational elements in the expression vector, which are homologous to the target locus are derived from a highly expressed locus meaning that they are derived from a gene, which is capable of high expression level in the host cell. A gene capable of high expression level, i.e. a highly expressed gene, is herein defined as a gene whose mRNA can make up at least 0.5% (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 1% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.1 g/l (as described in EP 357 127 B1). A number of preferred highly expressed fungal genes are given by way of example: the amylase, glucoamylase, alcohol dehydrogenase, xylanase, glyceraldehyde-phosphate dehydrogenase or cellobiohydrolase genes from Aspergilli or Trichoderma. Most preferred highly expressed genes for these purposes are a glucoamylase gene, preferably an A. niger glucoamylase gene, an A. oryzae TAKA-amylase gene, an A. nidulans gpdA gene, the locus of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29, preferably the A. niger locus of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29 or a Trichoderma reesei cellobiohydrolase gene.

[0125] Alternatively, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0126] Alternatively and/or in combination with the embodiments described above, the DNA construct comprising the reporter gene in operative association with the promoter of the invention may be deleted from the host cell after the cell has been screened for production, preferably secretion, of a protein of interest in step (e) of the invention. Step (e) is described later. This enables the host cell to be used directly for the production of the protein of interest, without said host cell comprising a reporter gene and/or selectable marker gene. To enable deletion of the DNA construct of the invention, the DNA construct preferably comprises both the reporter gene and a bi-directional selectable marker gene between DNA repeats. The DNA repeats allow deletion of the DNA construct by intra chromosomal homologous recombination. This method is analogous to the "MARKER-GENE FREE" approach as described in EP 0 635 574 B1.

[0127] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, Proceedings of the National Academy of Sciences USA 75:1433).

[0128] More than one copy of the DNA construct of the present invention may be inserted into the host cell. This can be done, preferably by integrating into its genome copies of the DNA sequence, more preferably by targeting the integration of the DNA sequence at a highly expressed locus, preferably at a glucoamylase locus or at the locus of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and SEQ ID NO:29. Alternatively, this can be done by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. To increase even more the number of copies of the DNA sequence to be over expressed the technique of gene conversion as described in WO98/46772 may be used.

[0129] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cell

[0130] The present invention further relates to recombinant host cells transformed with the DNA construct defined in the former section comprising a promoter DNA sequence of the present invention in operative association with a reporter gene. Preferably, the transformed host cell further comprises an additional DNA construct comprising a DNA sequence comprising a coding sequence originating from a DNA library from an organism suspected of being capable of producing one or more proteins with properties of interest.

[0131] Such cells can be advantageously used in an expression cloning method as described in the next section. An expression vector comprising a promoter DNA sequence of the present invention in operative association with a reporter gene is introduced into a host cell. The host cell is further transformed with a DNA construct or expression vector comprising a DNA sequence comprising a coding sequence originating from a DNA library. Both DNA construct or expression vectors are either maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier The choice of a host cell will to a large extent depend upon the organism wherein the DNA library has been made and/or upon the origin of the promoter DNA sequence used.

[0132] The host cell may be any microorganism. Preferably, the host cell is the same organism as the one wherein the DNA library has been prepared and/or as the one the promoter of the invention originates from as defined earlier in the specification. According to a preferred embodiment, the host cell is a prokaryote, or an eukaryote. According to a more preferred embodiment, the host cell useful in the methods of the present invention is a prokaryote, more preferably the prokaryote defined as source of promoter in the section promoter. More preferably, the prokaryotic host cell is a Bacillus host cell, preferably of the species Bacillus Subtilis, Bacillus licheniformis and/or Bacillus amyloliquefaciens.

[0133] According to another more preferred embodiment, the host cell useful in the methods of the present invention is a fungus.

[0134] The host cell may be a wild type filamentous fungus host cell or a variant, a mutant or a genetically modified filamentous fungus host cell. In a preferred embodiment of the invention the host cell is a protease deficient or protease minus strain. This may be the protease deficient strain Aspergillus oryzae JaL 125 having the alkaline protease gene named "alp" deleted (described in WO 97/35956 or EP 429 490), or the tripeptidyl-aminopeptidases (TPAP) deficient strain of A. niger, disclosed in WO 96/14404. Further, also host cell with reduced production of the transcriptional activator (prtT) as described in WO 01/68864 is contemplated according to the invention. Another specifically contemplated host strain is the Aspergillus oryzae BECh2, where the three TAKA amylase genes present in the parent strain IF04177 have been inactivated. In addition, two proteases, the alkaline protease and neutral metalloprotease 11 have been destroyed by gene disruption. The ability to form the metabolites cyclopiazonic acid and kojic acid has been destroyed by mutation. BECh2 is described in WO 00/39322 and is derived from JaL228 (described in WO 98/12300), which again was a mutant of IF04177 disclosed in U.S. Pat. No. 5,766,912 as A1560.

[0135] Optionally, the filamentous fungal host cell may comprise an elevated unfolded protein response (UPR) compared to the wild type cell to enhance production abilities of a polypeptide of interest. UPR may be increased by techniques described in US2004/0186070A1 and/or US2001/0034045A1 and/or WO01/72783A2 and/or WO2005/123763. More specifically, the protein level of HAC1 and/or IRE1 and/or PTC2 has been modulated, and/or the SEC61 protein has been engineered in order to obtain a host cell having an elevated UPR.

[0136] Alternatively, or in combination with an elevated UPR, the host cell may be genetically modified to obtain a phenotype displaying lower protease expression and/or protease secretion compared to the wild-type cell in order to enhance production abilities of a polypeptide of interest. Such phenotype may be obtained by deletion and/or modification and/or inactivation of a transcriptional regulator of expression of proteases. Such a transcriptional regulator is e.g. prtT. Lowering expression of proteases by modulation of prtT may be performed by techniques described in US2004/0191864A1 and in EP2005/055145.

[0137] Alternatively, or in combination with an elevated UPR and/or a phenotype displaying lower protease expression and/or protease secretion, the host cell may display an oxalate deficient phenotype in order to enhance the yield of production of a polypeptide of interest. An oxalate deficient phenotype may be obtained by techniques described in WO2004/070022A2 and/or WO2000/50576.

[0138] Alternatively, or in combination with an elevated UPR and/or a phenotype displaying lower protease expression and/or protease secretion and/or oxalate deficiency, the host cell may display a combination of phenotypic differences compared to the wild cell to enhance the yield of production of the polypeptide of interest. These differences may include, but are not limited to, lowered expression of glucoamylase and/or neutral alpha-amylase A and/or neutral alpha-amylase B, alpha-1, 6transglucosidase, protease, and oxalic acid hydrolase. Said phenotypic differences displayed by the host cell may be obtained by genetic modification according to the techniques described in US2004/0191864A1.

[0139] Alternatively, or in combination with phenotypes described here above, the efficiency of targeted integration of a nucleic acid construct into the genome of the host cell by homologous recombination, i.e. integration in a predetermined target locus, may preferably be increased by augmented homologous recombination abilities of the host cell. Such phenotype of the cell preferably involves a deficient hdfA or hdfB gene as described in WO2005/095624. WO2005/095624 discloses a preferred method to obtain a filamentous fungal cell comprising increased efficiency of targeted integration.

[0140] More preferably, the host cell of the invention is selected from the following list: a Bacillus, a yeast and a filamentous fungus, preferably an Aspergillus, Penicillium or Trichoderma species. Even more preferably, the Aspergillus host cell is an Aspergillus niger or Aspergillus sojae or Aspergillus oryzae species.

[0141] The present invention also relates to recombinant host cells, comprising more than one promoter DNA sequence of the present invention, each promoter being in operative association with a reporter gene. Such host cells may be advantageously used in an expression cloning method as described in the coming section. Bacterial cells may be transformed by a variety of methods including chemically induced competence (e.g. CaCl.sub.2) or electroporation (Sambrook et al. (1989) "Molecular Cloning: a laboratory manual", Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.), protoplast transformation (Chang and Cohen (1979) Mol Gen Genet. 168: 111) and, in the case of some Bacillus species, natural competence (Spizizen (1958) Proc Natl Acad Sci USA 44: 1072). Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable procedures for transformation of Aspergillus and other filamentous fungal host cells using Agrobacterium tumefaciens are described in e.g. Nat. Biotechnol. 1998 September; 16(9):839-42. Erratum in: Nat Biotechnol 1998 November; 16(11):1074. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. de Groot M J, Bundock P, Hooykaas P J, Beijersbergen A G. Unilever Research Laboratory Vlaardingen, The Netherlands. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156 and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920. Transformation of prokaryotes may be performed as described in EP 284 126A.

Method of Expression Cloning

[0142] According to a further aspect, the present invention provides a method for isolating a DNA sequence comprising a DNA sequence coding for a protein of interest in a host cell, said method comprises the steps of: [0143] (a) preparing a first DNA construct comprising a promoter DNA sequence operatively associated with a reporter gene conferring a selectable trait; said promoter DNA sequence being induced when the DNA construct is present in the host cell and when a protein of interest, preferably a secreted protein of interest, is produced by the host cell; [0144] (b) preparing a second DNA construct comprising a DNA sequence comprising a DNA sequence coding for a protein of interest originating from a DNA library from an organism suspected of being capable of producing one or more proteins of interest; [0145] (c) transforming a host cell with both DNA constructs prepared in (a) and (b); [0146] (d) culturing all the transformed host cells obtained in (c) under conditions conducive to the production of the proteins of interest as present in the DNA library; and [0147] (e) screening for transformed host cells producing a protein of interest by analysis of the proteins produced in (d).

Step (a)

[0148] A first DNA construct is prepared, comprising a promoter DNA sequence operatively associated with a reporter gene conferring a selectable trait; said promoter DNA sequence being induced when the DNA construct is present in the host cell and when a protein of interest, preferably a secreted protein of interest, is produced by the host cell.

[0149] The promoter present in the first DNA construct is a promoter which is induced when a protein of interest, preferably a secreted protein of interest, is produced by a host cell.

[0150] In the present invention the term "induced" is defined as an increase in promoter activity of a promoter of the invention when a protein of interest is (over)produced, preferably secreted, by a host cell, compared to the activity of the promoter when the protein of interest is not (over)produced in a corresponding control host cell under the same culture conditions. Preferably, when a protein of interest is (over)produced, preferably secreted, in a host cell, promoter activity is at least about 1.5-fold increased, more preferably at least about two-fold, more preferably at least about three-fold, more preferably at least about four-fold, more preferably at least about five-fold, even more preferably at least about six-fold, even more preferably at least about eight-fold, even more preferably at least about ten-fold, even more preferably at least about twenty-fold, even more preferably at least about 50-fold and most preferably at least about 100-fold increased, compared to a corresponding control host cell wherein the protein of interest is not (over)produced under the same culture conditions. More preferably, the increase in promoter activity is infinite, i.e. no promoter activity is detected or the promoter is inactive when the protein of interest is not (over)produced, whereas the promoter is induced when the protein of interest is (over)produced. Promoter activity is preferably determined as defined previously. Most preferably, promoter activity is determined by mRNA analysis using Northern blot.

[0151] Such a promoter can be identified by comparing gene expression profiles of cells producing the protein of interest with that of a control strain not producing said protein. Preferably, such expression profiles are compared using well-established DNA micro-array analyses. Alternatively, expression profiles can be compared using other methods known to the skilled person such as Northern blot or quantitative PCR analysis. A most preferred method to compare expression profiles is performed by first comparing expression profiles by DNA micro array analyses, followed by subsequent corroboration of the micro array results by Northern Blot. According to a preferred embodiment, the promoter is the promoter DNA sequence of the invention described under the section promoter.

[0152] According to another preferred embodiment, when the host cell is a Bacillus cell, the promoter is derived from the following genomic DNA sequences as listed: htrA (SEQ ID NO:1) or htrB (SEQ ID NO:2). Preferably, the promoter DNA sequence is derived from the non coding part of one of these genomic DNA sequences situated upstream of the start codon or part of said genomic sequences situated upstream. More preferably, the promoter used is the DNA sequence of either SEQ ID NO:1 or SEQ ID NO:2.

[0153] According to another preferred embodiment, when the host cell is a filamentous fungal cell, the promoter is derived from the following genomic DNA sequences as listed: SEQ ID NO:20, SEQ NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 24. Preferably, the promoter DNA sequence is derived from the non coding part of one of these genomic DNA sequences situated upstream of the start codon or part of said genomic sequences situated upstream. More preferably, the promoter used is the DNA sequence, or derivative thereof, of either SEQ ID NO:15, SEQ NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19.

[0154] The first DNA construct prepared in step (a) has already been described in the corresponding section "DNA construct" and preferably comprises a reporter gene conferring a selectable trait as described in the section "DNA construct".

Step, (b)

[0155] The DNA construct prepared in step (b) comprises a DNA sequence comprising a DNA sequence coding for a protein of interest originating from a DNA library from an organism suspected of being capable of producing one or more proteins of interest. The organism suspected of producing one or more proteins of interest usually is a prokaryote or an eukaryote. Examples of preferred prokaryotes and eukaryotes have already been defined earlier in the specification in connection with the origin of the promoter of the invention. According to one preferred embodiment, the organism is a Bacillus, more preferably a Bacillus species as defined in the section promoter. According to another preferred embodiment, the organism is an eukaryote, more preferably a fungus, of which most preferably a filamentous fungus.

[0156] The DNA construct prepared in step (b), preferably comprises regulatory sequences such as defined in the sections "DNA construct" and "Control elements".

[0157] In the method according to the invention, the library of DNA fragments from an organism suspected of producing one or more proteins of interest can be a genomic library or a cDNA library. However, in the case of eukaryotic donors, preferably a cDNA library is used so as to avoid problems with recognition of promoters or splice signals in the fungal host organism. The cDNA library is preferably prepared from mRNA isolated from the source organism when grown under conditions conducive to the expression of the proteins of interest.

[0158] The method according to the invention can be applied to the isolation of DNA sequences coding for any protein or polypeptide of interest if there is an assay available for detection of the protein when expressed by the host cell.

Step (c)

[0159] The identity of the host cell to be transformed with both DNA constructs has already been described under the section host cell. Methods of transformation have already been described in the same section. According to one embodiment, the host cell is simultaneously transformed with the constructs prepared in (a) and (b). Alternatively and according to another embodiment, the host cell is first transformed with the DNA construct prepared in step (a) and consecutively is transformed with the DNA construct prepared in step (b).

[0160] According to a preferred embodiment, the host cell is a prokaryote, preferably a Bacillus cell, more preferably a Bacillus subtilis, even more preferably a Bacillus subtilis deficient in the htrA gene, wherein at least part of the coding regions of the htrA gene have been deleted and/or replaced and/or wherein the non coding regions of the htrA gene are still present in the genome of said Bacillus cell, most preferably the Bacillus subtilis BV2003 is used (Hyyrylainen et al. (2001) Mol Microbiol 41: 1159).

[0161] Alternatively or in combination with former preferred embodiment, the host cell is a Bacillus cell and the reporter gene encodes a fluorescence reporter protein as defined in the section DNA construct, most preferably GFP.

[0162] Alternatively or in combination with former preferred embodiment, when the host cell is a Bacillus cell, the promoter is derived from the genomic DNA sequences as presented in the following list: htrA (SEQ ID NO:1) or htrB (SEQ ID NO:2). Preferably, the promoter sequence is derived from the non coding part of one of these two genomic DNA sequences situated upstream of the start codon or part of said sequence situated upstream. More preferably, the promoter used is one derived from either SEQ ID NO:1 or SEQ ID NO:2. According to a most preferred embodiment, the promoter used and the reporter gene used are either GFP in operative association with SEQ ID NO: 1 or GFP in operative association with SEQ ID NO: 2.

[0163] According to another preferred embodiment, the organism is an eukaryote, more preferably a fungus, of which most preferably a filamentous fungus. Preferably, the filamentous fungus is an Aspergillus, Penicillium or Trichoderma species. Even more preferably, the Aspergillus host cell is an Aspergillus niger or Aspergillus sojae or Aspergillus oryzae species. A most preferred host cell is A. niger, preferably CBS513.88 or derivative thereof.

[0164] Alternatively or in combination with former preferred embodiment, the host cell is a filamentous fungal cell and the reporter gene encodes a fluorescence reporter protein as defined in the section DNA construct, most preferably GFP. Alternatively, the reporter gene encodes a selectable marker. More preferably, the reporter gene is a hybrid reporter gene of a fluorescent protein and a selectable marker. Most preferably, the hybrid reporter gene is a comprises GFP and the phleomycin resistance gene (BLE) of Streptoalloteichus hindustanis (Drocourt et al., NAR, 1990).

[0165] Alternatively or in combination with the former preferred embodiment, when the host cell is a filamentous fungal cell, the promoter is derived from the genomic DNA sequences as presented in the following list: SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. Preferably, the promoter sequence is derived from the non coding part of one of the listed genomic DNA sequences situated upstream of the start codon or part of said sequence situated upstream. More preferably, the promoter used is one of those used in examples 3 to 7 and derived from either SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24. According to a most preferred embodiment, the promoter used and the hybrid reporter gene used are either GFP-BLE (SEQ ID NO: 61) in operative association with promoter SEQ ID NO: 15, GFP-BLE in operative association with promoter SEQ ID NO: 16, GFP-BLE in operative association with promoter SEQ ID NO: 17, GFP-BLE in operative association with promoter SEQ ID NO: 18, or GFP-BLE in operative association with promoter SEQ ID NO: 19.

Step (d)

[0166] After transformation of the host cells with both DNA constructs, all the obtained transformed host cells clones are cultured under conditions conducive to the production of the proteins of interest present in the DNA library. Depending on the assay required for detection of the protein of interest the transformed clones are propagated and stored as colonies on solid media such as agar plates or in liquid media, whereby the individual library clones are grown, stored and/or assayed in the wells of the microtiter plates.

[0167] The skilled person will understand that the usual adaptations to cloning methods known in the art can equally be applied to the method of the present invention. The adaptations include but are not limited to e.g. screening of pools of library clones, screening the same library for a number of different proteins of interest, as well as rescreening, reisolation and recloning of positive clones to ensure more accurate results.

[0168] A variety of methods are available to the skilled person for isolation of the DNA sequence encoding the protein of interest from the transformed host cell identified in the screening method, and for subsequent characterization of the isolated DNA sequence.

Step (e)

[0169] Subsequent to step (d), the transformed host cells are screened for production, preferably secretion, of a protein of interest by analysis of the proteins produced. During step (e), one screens for transformed host cells producing, preferably secreting a protein of interest, by monitoring production-dependent, preferably secretion-dependent reporter expression induced via the promoter DNA sequence present in the first DNA construct.

[0170] According to a preferred embodiment, when the host cell is a Bacillus cell, screening to identify transformed host cells producing, preferably secreting, a protein of interest by monitoring production-dependent, preferably secretion-dependent, reporter expression induced by the promoter DNA sequence present in the first DNA construct, is preferably performed using fluorescent based cell analysis assays, e.g. fluorescent activated cell scanning, fluorescent activated cell sorting (FACS) or fluorimetric analysis. More preferably, the host cell is a Bacillus cell comprising a first DNA construct, said DNA construct comprising a promoter such as SEQ ID NO: 1 or derivative thereof or SEQ ID NO: 2 or derivative thereof, said DNA construct further comprising a reporter gene encoding a fluorescent reporter and screening is performed using fluorescence based cell analysis assays. Even more preferably, the host cell is a Bacillus cell and the first DNA construct comprises: [0171] the promoter DNA sequence SEQ ID NO: 1 or derative thereof, in operative association with GFP, or [0172] the promoter DNA sequence SEQ ID NO: 2 or derative thereof, in operative association with GFP

[0173] and screening is performed using FACS. Alternatively, or in combination with the former embodiment, screening may be performed using e.g. colorimetric assays or using selective culture conditions. The skilled person will understand that depending on the reporter gene used, the selective culture conditions should be chosen accordingly. Examples of selective culture conditions are, but are not limited to, the use of biocides, the use of antibiotics, limitation of at least one nutrient, prototrophy.

[0174] According to another preferred embodiment, when the host cell is a filamentous fungal cell, screening to identify transformed host cells producing, preferably secreting, a protein of interest by monitoring production-dependent, preferably secretion-dependent, reporter expression induced by the promoter DNA sequence present in the first DNA construct of the invention, is preferably performed by cultivation under selective culture conditions. The skilled person will understand that depending on the reporter gene used, the selective culture conditions should be chosen accordingly. Examples of selective culture conditions are, but are not limited to, the use of biocides, the use of antibiotics, limitation of at least one nutrient, prototrophy. Alternatively, or in combination with the former embodiment, screening may be performed using e.g. colorimetric, fluorimetric (like FACS) or enzyme activity based assays. According to a more preferred embodiment, the host cell is a filamentous fungal cell comprising a first DNA construct of the invention and screening is performed using selective culture conditions. More preferably, the host cell is a filamentous fungal cell comprising a first DNA construct of the invention, said DNA construct comprising a promoter selected from the list of: SEQ ID NO:15 or derivative thereof, SEQ ID NO:16 or derivative thereof, SEQ ID NO:17 or derivative thereof, SEQ ID NO:18 or derivative thereof, SEQ ID NO:19 or derivative thereof, said DNA construct further comprising a reporter gene encoding a selectable marker and screening is performed using selective culture conditions. Even more preferably, the host cell is a filamentous fungal cell comprising a first DNA construct of the invention, said DNA construct comprising a promoter selected from the list of: SEQ ID NO:15 or derivative thereof, SEQ ID NO:16 or derivative thereof, SEQ ID NO:17 or derivative thereof, SEQ ID NO:18 or derivative thereof, SEQ ID NO:19 or derivative thereof, said DNA construct further comprising a hybrid reporter gene such as SEQ ID NO: 61 and screening is performed using selective culture conditions.

[0175] According to another more preferred embodiment, the host cell is an A. niger cell comprising a first DNA construct of the invention and screening is performed using selective culture conditions. More preferably, the host cell is an A. niger cell comprising a first DNA construct of the invention, said DNA construct comprising a promoter selected from the list of: SEQ ID NO:15 or derivative thereof, SEQ ID NO:16 or derivative thereof, SEQ ID NO:17 or derivative thereof, SEQ ID NO:18 or derivative thereof, SEQ ID NO:19 or derivative thereof, said DNA construct further comprising a reporter gene encoding a selectable marker and screening is performed using selective culture conditions. Even more preferably, the host cell is an A. niger cell comprising a first DNA construct of the invention, said DNA construct comprising a promoter selected from the list of: SEQ ID NO:15 or derivative thereof, SEQ ID NO:16 or derivative thereof, SEQ ID NO:17 or derivative thereof, SEQ ID NO:18 or derivative thereof, SEQ ID NO:19 or derivative thereof, said DNA construct further comprising a hybrid reporter gene such as SEQ ID NO:61 and screening is performed using selective culture conditions. Even more preferably, the host cell is an A. niger cell and the first DNA construct comprises: [0176] hybrid reporter gene GFP-BLE (SEQ ID NO: 61) in operative association with promoter SEQ ID NO: 15, or [0177] GFP-BLE in operative association with promoter SEQ ID NO: 16, or derivative thereof, or [0178] GFP-BLE in operative association with promoter SEQ ID NO: 17, or derivative thereof, or [0179] GFP-BLE in operative association with promoter SEQ ID NO: 18, or derivative thereof, or [0180] GFP-BLE in operative association with promoter SEQ ID NO: 19, or derivative thereof, and screening is performed using selective culture conditions comprising an antibiotic, preferably phleomycin as selective agent.

[0181] The DNA sequences isolated by the screening method of the invention as described above are used to produce, or to improve the production of, a protein of interest encoded by the DNA sequence. Advantageously, the transformed host cell as isolated in the above described screening method is used directly in a process for the production of the protein of interest by culturing the transformed host cell under conditions conducive to the production, preferably secretion of the protein of interest and, optionally, recovering the protein.

[0182] According to a preferred embodiment, the host cell is enabled to be used directly for the production of the protein of interest, without said host cell comprising a reporter gene and/or selectable marker gene. The DNA construct comprising the reporter gene in operative association with the promoter of the invention is therefore deleted from the host cell after the cell has been screened for production, preferably secretion, of a protein of interest. To enable deletion of the DNA construct of the invention, the DNA construct preferably comprises both the reporter gene and a bi-directional selectable marker gene between DNA repeats. The DNA repeats allow deletion of the DNA construct by intra chromosomal homologous recombination. This method is analogous to the "MARKER-GENE FREE" approach as described in EP 0 635 574 B1.

[0183] Often the initial transformed host cell isolated in the screening method of the invention will have an expression level which is satisfactory for screening purposes but which can be significantly improved for economic production purposes. To this end, the DNA sequence is inserted into an expression vector which is subsequently used to transform a suitable host cell. In the expression vector the DNA sequence is operably linked to appropriate expression signals, such as a promoter, optionally a signal sequence and a terminator, which are capable of directing the expression of the protein in the host organism as defined in the sections "DNA construct" and "control elements". A suitable host cell for the production of the protein may be either a prokaryotic or eukaryotic cell, preferably a eubacterium, yeast or filamentous fungus. Preferred bacterial host cells are selected from the genus Bacillus as defined in the section promoter. According to another preferred embodiment, the host cell is a yeast or a filamentous fungus as already defined in the section "host cell". Preferred yeast host cells are selected from the group consisting the genera Saccharomyces, Kluyveromyces, Yarrowia, Pichia, and Hansenula. Preferred filamentous fungal host cells are selected from the same genera listed above as preferred host cells for the screening method. More preferably, the filamentous fungus is an Aspergillus, Penicillium or Trichoderma species. Even more preferably, the Aspergillus host cell is an Aspergillus niger or Aspergillus sojae or Aspergillus oryzae species. A most preferred host cell is A. niger, preferably CBS513.88 or derivative thereof.

[0184] The suitable host cell is transformed with the expression vector by any of the various protocols available to the skilled person. The transformed host cell is subsequently used in a process for producing the protein of interest by culturing the transformed host cell under conditions conducive to the expression of the DNA sequence encoding the protein, and optionally recovering the protein.

[0185] Preferably, the protein of interest is an enzyme.

[0186] Accordingly, the invention relates to a use of the promoter of the invention in an expression cloning method.

[0187] The invention further relates to other use of the promoter of the invention. Preferably, the promoter of the invention may be operatively associated with any coding sequence in a DNA construct or expression vector all defined in former sections. Such DNA contruct or expression vector may be introduced into any host cell as defined in "host cell" section and used for expression of a given valuable compound. Examples of such valuable compound are, but are not limited to, a metabolite or a polypeptide. Preferably, the coding sequence operatively associated with the promoter of the invention encodes the valuable compound. Alternatively, and according to another preferred embodiment of the invention, the coding sequence operatively associated with the promoter of the invention is involved in the production of the valuable compound.

[0188] Alternatively and according to another preferred use of the promoter of the invention, the promoter of the present invention is incorporated into an inactivation or replacement construct, transformed into a given host cell and used to inactivate or replace a target gene.

[0189] The present invention is further illustrated by the following examples.

EXAMPLES

Materials and Methods

General Procedures

[0190] Standard molecular cloning techniques such as DNA isolation, gel electrophoresis, enzymatic restriction modifications of nucleic acids, Northern analyses, E. coli transformation, etc, were performed as described by Sambrook et al. (2001) "Molecular Cloning: a laboratory manual, 3.sup.rd edition", Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y. and Innis et al. (1990) "PCR protocols, a guide to methods and applications" Academic Press, San Diego. Synthetic oligo deoxynucleotides were obtained from Invitrogen (Breda, The Netherlands). Room temperature is 20 degrees Celsius.+-.2 degrees Celsius.

Transformation of Aspergillus niger.

[0191] Transformation of A. niger was performed according to the method described by Tilburn, J. et al. (1983) Gene 26, 205-221 and Kelly, J. & Hynes, M. (1985) EMBO J., 4, 475-479 with the following modifications:

[0192] Spores were germinated and cultivated for 16 hours at 30 degrees Celsius in a shake flask placed in a rotary shaker at 300 rpm in Aspergillus minimal medium (100 ml). Aspergillus minimal medium contains per litre: 6 g NaNO.sub.3, 0.52 g KCl, 1.52 g KH.sub.2PO.sub.4, 1.12 ml 4 M KOH, 0.52 g MgSO.sub.4.7H.sub.2O, 10 g glucose, 1 g casaminoacids, 22 mg ZnSO.sub.4.7H.sub.2O, 11 mg H.sub.3BO.sub.3, 5 mg FeSO.sub.4.7H.sub.2O, 1.7 mg CoCl.sub.2.6H.sub.2O, 1.6 mg CuSO.sub.4.5H.sub.2O, 5 mg MnCl.sub.2.2H.sub.2O, 1.5 mg Na.sub.2MoO.sub.4.2H.sub.2O, 50 mg EDTA, 2 mg riboflavin, 2 mg thiamine-HCl, 2 mg nicotinamide, 1 mg pyridoxine-HCL, 0.2 mg panthotenic acid, 4 g biotin, 10 ml Penicillin (5000 IU/ml) Streptomycin (5000 UG/ml) solution (Gibco). [0193] Novozym 234.TM. (Novo Industries) instead of helicase was used for the preparation of protoplasts; [0194] after protoplast formation (60-90 minutes), KC buffer (0.8 M KCl, 9.5 mM citric acid, pH 6.2) was added to a final volume of 45 ml, the protoplast suspension was centrifuged for 160 minutes at 3000 rpm at 4 degrees Celsius in a swinging-bucket rotor. The protoplasts were resuspended in 20 ml KC buffer and subsequently 25 ml of STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl.sub.2) was added. The protoplast suspension was centrifuged for 10 minutes at 3000 rpm at 4 degrees Celsius in a swinging-bucket rotor, washed in STC-buffer and resuspended in STC-buffer at a concentration of 10E8 protoplasts/ml; [0195] to 200 microliter of the protoplast suspension, the DNA fragment, dissolved in 10 microliter TE buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) and 100 microliter of PEG solution (20% PEG 4000 (Merck), 0.8 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl.sub.2) was added; [0196] after incubation of the DNA-protoplast suspension for 10 minutes at room temperature, 1.5 ml PEG solution (60% PEG 4000 (Merck), 10 mM Tris-HCl pH 7.5, 50 mM CaCl.sub.2) was added slowly, with repeated mixing of the tubes. After incubation for 20 minutes at room temperature, suspensions were diluted with 5 ml 1.2 M sorbitol, mixed by inversion and centrifuged for 10 minutes at 4000 rpm at room temperature. The protoplasts were resuspended gently in 1 ml 1.2 M sorbitol and plated onto solid selective regeneration medium consisting of either Aspergillus minimal medium without riboflavin, thiamine.HCL, nicotinamide, pyridoxine, panthotenic acid, biotin, casaminoacids and glucose. In case of acetamide selection the medium contained 10 mM acetamide as the sole nitrogen source and 1 M sucrose as osmoticum and C-source. Alternatively, protoplasts were plated onto PDA (Potato Dextrose Agar, Oxoid) supplemented with 1-50 microgram/ml phleomycin and 1 M sucrose as osmosticum. Regeneration plates were solidified using 2% agar (agar No. 1, Oxoid L11). After incubation for 6-10 days at 30 degrees Celsius, conidiospores of transformants were transferred to plates consisting of Aspergillus selective medium (minimal medium containing acetamide as sole nitrogen source in the case of acetamide selection or PDA supplemented with 1-50 microgram/ml phleomycin in the case of phleomycin selection) with 2% glucose and 1.5% agarose (Invitrogen) and incubated for 5-10 days at 30 degrees Celsius. Single transformants were isolated and this selective purification step was repeated once upon which purified transformants were stored. Aspergillus niger Microtiterplate Fermentation and Sampling

[0197] To obtain spores of A. niger, mycelium was transferred to microtiterplates filled with 150 microliter solid PDA medium (Potato Dextrose Agar, Oxoid), prepared according to the supplier's instructions. After growth for 3-7 days at 30 degrees Celsius (static) spores had formed. Subsequently 100 microliter liquid fermentation medium (70 g/l glucose.H2O, 25 g/l peptone (from casein), 12.5 g/l yeast extract (Difco), 2 g/l K.sub.2SO.sub.4, 1 g/l KH.sub.2PO.sub.4, 0.5 g/l MgSO.sub.4.7H.sub.2O, 0.03 g/l ZnCL.sub.2, 0.02 g/l CaCL.sub.2, 9 mg/l MnSO.sub.4.H.sub.2O, 3 mg/l FeSO.sub.4.7H.sub.2O, adjusted to pH 5.6 with 4 NH.sub.2SO.sub.4), was added to each well. The fermentation medium was pipetted up and down 20 times to isolate the spores in the medium. The inoculated fermentation medium was transferred to another microtiterplate and incubated at 34 degrees Celsius, 550 rpm and 80% humidity for 6 days in an orbital microtiterplate shaker (Infors HT). On day 6 supernatants were collected and used for detection of secreted proteins.

Mass Spectrometry Analysis

[0198] Supernatants were ultra-filtrated by centrifugal force on an Ultracel filtration plate (Millipore, Billerica, Mass., USA). Ultra-filtration was performed according to the protocol provided by the manufacturer. To the ultra-filtrated supernatants 10 microliter 200 mM ammoniumbicarbonate, pH 7,8 (Riedel-de Haen A G, Seelze, Germany) and 1 microliter 250 microgram per ml trypsin (Worthington, Lakewood, N.J., USA) were added. The supernatants were digested for 3 hours at 37 degrees Celsius. The digested supernatants were mixed in a 1:1 ratio with 10 milligram per ml recrystallized alpha-cyano-4-hydroxycinnamic acid (?CHCA) (Laser Bio Labs, Sophia-Antipolis Cedex, France) solution; from these mixtures 1 microliter was spotted on the MALDI target. The spotted samples were analyzed with MALDI MS and MALDI MS/MS on a vMALDI LTQ mass spectrometer (Thermo electron, Orlando, Fla., USA).

[0199] The obtained MS/MS spectra, converted to peptide sequences, were compared to available protein sequences in pre-constructed FASTA protein databases, to identify individual enzymes.

RNA Isolation

[0200] Aspergillus niger mycelium formed on 20 ml liquid selective regeneration medium (6 g/l NaNO.sub.3, 0.52 g/l KCl, 1.52 g/l KH.sub.2PO.sub.4, 0.25 g/l KOH, 0.52 g/l MgSO.sub.4.7H.sub.2O, 22 mg/l ZnSO.sub.4.7H.sub.2O, 11 mg/l H.sub.3BO.sub.3, 5 mg/l FeSO.sub.4.7H.sub.2O, 1.7 mg/l CoCl.sub.2.6H.sub.2O, 1.6 mg/l CuSO.sub.4.5H.sub.2O, 5 mg/l MnCl.sub.2.2H.sub.2O, 1.5 mg/l Na.sub.2MoO.sub.4.2H.sub.2O, 50 mg/l EDTA, 341 g/l sucrose, 10 ml Penicillin (5000 IU/ml) Streptocmycin (5000 UG/ml) solution (Gibco)) in a petridish was harvested, washed with demineralized water and squeezed between paper towels to remove excessive water. Mycelium was frozen immediately in liquid nitrogen and grinded to a fine powder using mortar and pestle. The resulting powder was transferred to a sterile 50 ml tube and weighed upon which for every 1-1.2 g of ground mycelium 10 ml TRIzol reagent (Invitrogen) was added (max. 25 ml per tube). The mycelial powder was immediately solubilized by vigorous mixing (vortexing, 1 min.), followed by 5 min room temperature incubation with occasional mixing. 0.2 (original TRIzol) volumes of chloroform (thus 2 ml for every 10 ml TRIzol used originally) was added, vortexed and left at room temperature for 10 min. Subsequently, the mixture was centrifuged at 4 degrees Celsius, 6000 g for 30 minutes. The top aqueous phase was transferred to a fresh tube and total RNA was precipitated by addition of 0.5 (original TRIzol) volumes of 100% isopropyl alcohol (thus 5 ml of isopropyl alcohol for every 10 ml TRIzol used originally). After 10 minutes precipitation at room temperature, the RNA was recovered by centrifugation for 30 minutes at 6000 g. Upon removal of supernatant, the RNA pellet was rinsed with one (original TRIzol) volume of 70% ethanol. After removal of the ethanol, the RNA pellet was air dried. The dried RNA pellet was dissolved in 3 ml GTS (100 mM Tris-Cl, pH 7.5, 4 M guanidium thiocyanate, 0.5% sodium lauryl sarcosinate) buffer. Additional clean-up of the RNA was performed using Rneasy Maxi Kit (Qiagen) according to suppliers manual. 10 microliter of RNA solution was used to determine quality and concentration of nucleic acids.

Aspergillus niger Strains

[0201] A. niger WT-1: This A. niger strain is CBS513.88 comprising deletions of the genes encoding glucoamylase (glaA), fungal amylase and acid amylase. A. niger WT 1 was constructed by using the "MARKER-GENE FREE" approach as described in EP 0 635 574 B1. In this patent it is extensively described how to delete glaA specific DNA sequences in the genome of CBS 513.88. The first procedure resulted in a MARKER-GENE FREE ?glaA recombinant A. niger CBS 513.88 strain, possessing finally no foreign DNA sequences at all. Subsequently, the genes encoding fungal amylase and acid amylase were deleted from the genome of A. niger WT-1 by the method described in EP 0 635 574 B1. Vectors used for cloning of the desired constructs as depicted in FIGS. 7 to 9 and 11 to 14 contain backbone features previously described in WO 99/32617.

A. niger WT-GFP: This A. niger strain was obtained by transformation of A. niger WT-1 with the expression vector pGBFINGFP-2 depicted in FIG. 7 containing the gene coding for the well-known Green Fluorescent Protein (GFP))(Chalfie, M et al., Science (1994) 263(5148): 802-805) driven by the glucoamylase promoter. After purification on selective medium, a single copy integrant was selected based on PCR analysis. A. niger WT-PHY: This A. niger strain was obtained by transformation of the A. niger WT-1 with the expression vector pGBFIN-32 depicted in FIG. 8 containing the phytase gene PHY (identical to FytA previously described in WO 99/32617) driven by the glucoamylase promoter. After purification on selective medium, a single copy integrant was selected based on PCR analysis.

[0202] A. niger WT-PHY-2: This A. niger strain was obtained by transformation of the A. niger WT-1 with the expression vector pGBFIN-32 depicted in FIG. 8 containing the phytase gene PHY (identical to FytA previously described in WO 99/32617) driven by the glucoamylase promoter. After purification on selective medium, a multy copy integrant was selected based on PCR analysis.

[0203] A. niger WT-vector: This A. niger strain was obtained by transformation of the A. niger WT-1 strain with empty control vector pGBFIN-40, depicted in FIG. 9. The empty control vector pGBFIN-40 was constructed by XhoI digestion of pGBFIN-32 depicted in FIG. 8, and re-ligation of the largest fragment, thereby removing the glucoamylase promoter and the 5' part of phytase gene PHY. A single copy integrant was selected by PCR analysis.

Example 1

Construction of Bacillus Reporter Plasmids and Reporter Strains

[0204] 1.1 Construction of Reporter Plasmids pPhtrA-gfp-amyE and pPhtrB-gfp-amyE

[0205] First, reporter plasmid pPhtrA-gfp-amyE was constructed as follows. The gene encoding the optimized version of GFP, gfpmut-1 (P. Cormack, R. H. Valdivia, and S. Falkow, 1996; FACS-optimized mutants of the green fluorescent protein (GFP), Gene 173, 33.), was amplified by PCR, using pSG1151 (P. J. Lewis and A. L. Marston, 1999; GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis, Gene 227, 101.) as template DNA, with the following primers: RN-lacZ-rv (SEQ ID NO: 3), GTGAGCGGATGCAATTTCACACAGG; and gfp-terminator-rv, (SEQ ID NO: 4) GTGGCTCAGCTTTTTTAAGGAAGGGAGGCTCTCACCTCCCTTCCCTTTATTTGTAGAGC TCATCCATGCC, resulting in KpnI and Bpu1101I (in bold) sites up- and downstream gfpmut-1, respectively. The 913 bp PCR product was ligated in pDL (containing homologous flanking regions of the amyE locus for site specific recombination, (G. Yuan and S. L. Wong, 1995; Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE), J. Bacteriol. 177, 5427), using the KpnI and Bpu11011 sites, resulting in pGFP-amyE. The promoter of htrA (P.sub.htrA) (SEQ ID NO: 1) was amplified by PCR using chromosomal DNA of B. subtilis 168 (Bacillus Genetic Stock Center ID: 1A1) as template DNA. Primers used were: PhtrA-fw, (SEQ ID NO: 5) CGTGAGGTACCGGCTTCTGTTTCTGCC; and Phtr-rv, (SEQ ID NO: 6) CATCACGAAGCTTATCCATCATGTTCACTCCG, introducing Kpn1 and HindIII sites (in bold), respectively. After digestion with Kpn1 and Hind3, the 531 bp PCR product was ligated into pGFP-amyE, upstream the gfp gene resulting in reporter plasmid pPhtrA-gfp-amyE. A map of this plasmid is depicted in FIG. 1. Plasmid pPhtrB-gfp-AmyE was constructed in a similar way as described above. The primers used for the amplification of the promoter of htrB (P.sub.htrB) (SEQ ID NO 2) were PhtrB-fw (SEQ ID NO 7) GACCGGTACCTCAGGATCTTTCGCC and PhtrA-rv (SEQ ID NO 8) GGCCATCAAGCTTATAATCCATGTTCTTACACTCC.

1.2 Construction of the Bacillus Reporter Strains VT210A and VT210B

[0206] The resulting plasmids pPhtrA-gfp-amyE and pPhtrB-gfp-amyE were introduced into B. subtilis DB104, a DaprE, DnprE double mutant (F. Kawamura and R. H. Doi, 1984; Construction of a Bacillus subtilis double mutant deficient in extra cellular alkaline and neutral proteases, J. Bacteriol. 160, 442). Chloramphenicol resistant transformants were checked for site-specific integration by double crossover at the amyE locus by PCR on chromosomal DNA. To reduce negative feedback regulation by HtrA of P.sub.htA, (D. Noone, A. Howell, R. Collery, and K. M. Devine, 2001; YkdA and YvtA, HtrA-like serine proteases in Bacillus subtilis, engage in negative auto regulation and reciprocal cross-regulation of ykdA and yvtA gene expression, J. Bacteriol. 183, 654) the new strain was transformed with chromosomal DNA of B. subtilis BV2003, which has a disrupted htrA gene by integration of pMutin2 (H. L. Hyyrylainen, A. Bolhuis, E. Darmon, L. Muukkonen, P. Koski, M. Vitikainen, M. Sarvas, Z. Pragai, S. Bron, J. M. van Dijl, and V. P. Kontinen, 2001; A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress, Mol. Microbiol. 41, 1159). Transformants were selected for erythromycin resistance and checked by PCR on chromosomal DNA for htrA disruption. The obtained strains were designated VT200A and VT200B.

[0207] In order to increase transformation efficiency of the VT200A strain, a BsuMR disruption construct was designed. The BsuMR system is formed by an operon consisting of three genes, ydiR, ydiS and ydjA, of which each is essential for BsuMR functioning (P. J. Lewis and A. L. Marston, 1999; GFP vectors for controlled expression and dual labeling of protein fusions in Bacillus subtilis, Gene 227,101). Flanking region 1 (flr1), a 756 bp region of ydiR was amplified using the following primers: flrBsu1F, (SEQ ID NO 9) GAAAGATTGTTTCAGAAGCC; and frl1 BsuR, (SEQ ID NO 10) ACAGCGTTGGGATCCAAGCCCTTCCATTTTGGACATTTGG and chromosomal DNA of B. subtilis as a template. The spectinomycin resistance marker was amplified using pDG1726 (A. M. Guerout-Fleury, K. Shazand, N. Frandsen, and P. Stragier, 1995; Antibiotic-resistance cassettes for Bacillus subtilis, Gene 167, 335) as template DNA and with the following primers: specBsu-F, (SEQ ID NO 11) GGGCTTGGATCCCAACGCTGTCGACGTTGTAAAACGACGG; and specBsu-R, (SEQ ID NO 12) CGCATAGCTTTCCGGTCGCCGCAGCTATGACCATGATTACGC. The 1319 bp PCR product and the flrBsu1 fragment were used in a second PCR in which both fragments served as one template upon recombination of their homologous ends introduced by primers flrBsu1-R and specBsu-F (in bold). Using primers flrBsu1-F and specBsu-R, a 2075 bp PCR fragment, flr1-spec.sup.r, was obtained, which was sub cloned in pCR-XL-TOPO (Invitrogen) resulting in pCT-XL-TOPO-flr1-spec. Flanking region 2, a 739 bp region covering 24 bp of the 3' end of ydiS (encoding a DNA endonuclease) and a large part of the adjacent ydjA (unknown function), was amplified using primer flrBsu2-F, (SEQ ID NO 13) GATGATTCGTCTTTTTGTAGTG; and primer flrBsu2-R, (SEQ ID NO 14) CGATCAACATATGTGTTCACG and chromosomal DNA of B. subtilis as a template. The PCR product was cloned in pCR-XL-TOPO resulting in pCR-XL-TOPO-flr2. Making use of the multiple cloning site of the pCR-XL-TOPO vector, flr1-spec.sup.r was restricted from pCR-XL-TOPO-flr1 using XhoI and NsiI and ligated into pCR-XL-TOPO-flr2, digested with XhoI and PstI (compatible to NsiI). This resulted in the deletion construct pTOPO-flr1-spec.sup.r-flr2, which was introduced into strain VT200A. Spectinomycin resistant transformants were checked for the ydiS deletion by PCR on chromosomal DNA. The obtained, final reporter strains were designated VT210A and VT210B and used for further experiments. A general outline of the construction of these strains is depicted in FIG. 2.

1.3 Construction of Expression Vectors pUBnpr2 and pUBBAG:

[0208] The construction of plasmid pUB110 and its sequence was described before (McKenzie T., Hoshino T., and Sueoka, N. 1986. The nucleotide sequence of pUB110; some salient features in relation to replication and its regulation. Plasmid 15: 93-103). Cloning of the Bacillus amyloliquefaciens a-amylase gene amyQ (Genbank accession number J 01542) in pUB110 lead to plasmid pKTH10 as described before (Palva, I. 1982. Molecular cloning of the alpha-amylase gene of Bacillus amyloliquefaciens and its expression in B. subtilis. Gene 19: 81-87).

[0209] Cloning of the neutral protease gene npr (Genbank accession number K 02497) and .beta.-glucanase gene bag (Genbank accession number M15674) both from Bacillus amyloliquefaciens into vector pUB110 was performed essentially according to the method used for the cloning of amyQ, resulting in plasmids pUBnpr2 and pUBBAG as depicted in FIGS. 3 and 4.

1.4 Transformation of Expression Constructs pKTH10, pUBnpr2 and pUBBAG into Bacillus Reporter Strain VT210A:

[0210] Transformation of Bacillus subtilis strain VT210A was essentially performed as described before making use of the natural competence of this organism (Anagnostopoulos, C. and Spizizen, J. 1961. Requirements for transformation in Bacillus subtilis. J. Bacteriology 81: 741-746). After DNA incubation the cells were spread on 2.times.TY agar containing 0.1% starch and the antibiotic kanamycin (20 .mu.g/ml) as a selective agent for transformation. Plates were incubated over night at 37.degree. C. When plasmid pKTH10 was involved in transformation the colonies were covered with a lugol solution to detect the amylase secreting cells.

Example 2

Analysis of Secretion Stressed Cells by Fluorescent Activated Cell Assays

2.1 Selection of Secretion Stressed Cells by Fluorescence Activated Cell Sorting:

[0211] The plasmids pUB110 (empty control vector) and pKTH10 (encoding Bacillus amyloliquefaciens a-amylase gene amyQ) were used in a ratio of 1:20 for co-transformation of Bacillus subtilis strain VT210A. One part of the cells was spread on selective TY agar plates, and to the other part 2.times.TY broth containing kanamycin, was added and the cells were cultivated over night. The separate plasmids were transformed as well, performing the same operations as for the co-transformation. The next day, cultures were analyzed for GFP fluorescence on a flow cytometer type FACS (Mo-Flo of Dako-cytomation) by a skilled operator. A blue laser of 80 mWatt, and 488 nm was used. A typical FACS experiment comprised analysis of 20.000 events (cells). First, fluorescence signals of cells of VT210A, pUB110 (no secretion stress) and of VT210A, pKTH10 (secretion stress due to overproduction of the alpha-amylase AmyQ) were analyzed separately (FIGS. 5A and 5B) and as a mix (FIG. 5C). On basis of these results a cell-sorting limit was manually set (dashed line in FIG. 5D), such that cells demonstrating GFP fluorescence higher than the cell-sorting limit, were sorted. The co-transformation culture was applied to the flow cytometer and sorted cells were collected in a tube. To verify that the sorted fluorescent population had been enriched for alpha-amylase secreting cells, different dilutions of the collected cells were spread on selective agar containing starch. The same was done with the original overnight input culture, which was not sorted by FACS. After growth, the colonies were covered by a lugol solution to visualize halo formation caused by alpha-amylase activity. As shown in table 1, about 90% of the sorted cells indeed secreted alpha-amylase, whereas in the input culture only 0.5-0.7% of the cells secreted alpha-amylase. This result clearly demonstrates the powerful selection tool of the present invention by isolation of protein secreting cells using discrimination in fluorescent signal between normal cells and cells stressed by secretion.

TABLE-US-00001 TABLE 1 enrichment for amy+ colony forming units after flow cytometry. Number Number Used cells of cfu of amy.sup.+ Dilution % amy.sup.+ Pre-FACS of over night >2000 14 10.sup.-4 <0.7 grown co-transformation 600 3 10.sup.-5 0.5 Sorted cells of over night 111 104 10.sup.-1 93.7 grown co- transformation 9 8 10.sup.-2 88.9

2.2 Selection of Secretion Stressed Cells by Fluorescent Cell Scanning:

[0212] The plasmids pUB110 (empty control vector) and pKTH10 (encoding Bacillus amyloliquefaciens a-amylase gene amyQ) were transformed separately to Bacillus subtilis strain VT210B. One part of the cells was spread on selective TY agar plates, and to the other part 2.times.TY broth containing kanamycin, was added and the cells were cultivated over night. Cultures were analysed by a skilled operator on a flow cytometer (Coulter Epics XL-MCL, Beckman Coulter). A typical experiment comprised analysis of 20.000 events (cells). Fluorescent signal was detected using a FITC-filter and setting of the photo multiplier voltage was between 700 and 800 Volts. The difference in fluorescent signal between the normal cells and cells stressed by secretion is depicted in FIG. 6.

2.3 Selection for Secretion Stressed VT210A Cells Using a Fluorimeter:

[0213] Transformed reporter strain cells were inoculated in 2.times.TY broth in the presence of kanamycin and grown over night at 37.degree. C. The DNA's used for transformation were equal amounts of the plasmids pUB110, pKTH10, pUBnpr2 and pUBBAG. After growth to stationary phase the cultured cells of the four transformations were divided 8-fold in equal volumes in a 96 well micro titer plate format. The wells were analyzed for fluorescence in a fluorimeter (Molecular Devices, type Spectra MAX Gemini). Excitation is at 490 nm, emission at 510 nm, and cutoff at 495 nm. A standard t-Test assuming unequal variances was performed. Table 2 demonstrates that non-stressed cells (pUB110) were distinguished from the stressed cells (PKTH10, pUBnpr2 and PUBBAG) by a clear significant (P two-tail<0.05) difference in fluorescent signal.

TABLE-US-00002 TABLE 2 t-Test assuming unequal variances. pUB110 cells Ratio pKTH10 cells P = 1.05899E-13 7.84 pUBnpr2 cells P = 4.63415E-11 1.58 pUBBAG cells P = 7.17216E-14 1.77 P-values are given for empty host cells (pUB110) versus secretion stressed (pKTH10, pUBnpr2, pUBBAG) cells. The ratio expresses the mean (n = 8) fluorescent value of stressed cells over the mean (n = 8) fluorescent value of empty host cells.

Example 3

Genome-Wide Expression Analyses of A. niger to Identify Secretion-Induced Promoters

[0214] Genome-wide expression analyses were performed to identify promoter sequences that are highly expressed in A. niger WT-PHY, and show no or low expression in A. niger WT-GFP, A. niger WT-vector and A. niger WT-1. To simulate future application experiments protoplasts were obtained of A. niger WT-1, A. niger WT-GFP, A. niger WT-PHY and A. niger WT-vector strains. An amount of 20 ml liquid selective regeneration medium supplemented with 50 mg/l phleomycin (Invitrogen) in case of A. niger WT-GFP, A. niger WT-PHY and A. niger WT-vector, was inoculated in a petri dish with 100 microliter 10E7/ml protoplasts. After a growth period of 3 days at 30 degrees Celsius (static), a mycelial pellet had formed at the surface of the liquid medium which was used for total RNA isolation. cDNA synthesis, labelling of the cDNA and hybridisation on Affymetrix A. niger GeneChips.TM. was performed according to suppliers protocol. Subsequent expression analysis resulted in identification of 5 genes that had an expression level in the A. niger WT-PHY strain of at least 8.5 times the basal expression level in A. niger WT-1, A. niger WT-GFP and A. niger WT-vector. To confirm the findings, Northern blot analysis was performed.

Example 4

Northern Analysis

[0215] To corroborate the results of the Affymetrix A. niger GeneChips.TM. experiments, Northern blot analysis was performed. For this, total RNA was isolated from A. niger strains WT-1, WT-GFP, WT-FYT and WT-vector. RNA was denatured, separated by gel electrophoresis on a 1% agarose gel and transferred onto a nylon membrane (Hybond-N.sub.+, Amersham Biosciences) by capillary blotting according to the manufacturer's instruction. The RNA was UV cross-linked to the nylon membranes on a GS Gene Linker.TM. (BioRad, C3 setting, 150 mJoule). DNA for the generation of .sup.32P-labeled DNA probes was isolated by PCR using genomic DNA of A. niger WTF-1 as a template and using the primers listed in Table 3 (SEQ ID NO's 50 to 59). Northern blots were hybridised with randomly primed .sup.32P-labeled DNA probes, representing the secretion-induced genes (SEQ ID NO's 20 to 24), according to suppliers instructions (RadPrime DNA Labeling System, Invitrogen). The results of the Northern analyses are shown in FIG. 10. The hybridisation pattern of the Northern blots confirm the data obtained by expression analyses of the Affymetrix A. niger GeneChips.TM. data; low expression of the secretion-induced genes in A. niger strains WT-1, WT-GFP, WT-vector and high expression in A. niger WT-PHY.

TABLE-US-00003 TABLE 3 Primers used to isolate DNA for 32P-labeled probes. Primer Primer sequence Gene sequence SEQ ID NO: 50 CAACACCTACGGCGTCAAGG SEQ ID NO:20 SEQ ID NO: 51 TAACCCGGGGAGATGCTGTT SEQ ID NO:20 SEQ ID NO: 52 CGATCCGGAAATTCCTCCTG SEQ ID NO:21 SEQ ID NO: 53 GCCGCCGACTCAGCAGTAT SEQ ID NO:21 SEQ ID NO: 54 GTGCATGAACAACACGGAGA SEQ ID NO:22 SEQ ID NO: 55 ACTCAGTVGTGCGCCTCTCG SEQ ID NO:22 SEQ ID NO: 56 GTCGAACTCGGCGTVTCTCC SEQ ID NO:23 SEQ ID NO: 57 CCGGTGAGGAGGTGCTTVTC SEQ ID NO:23 SEQ ID NO: 58 AGCATCTCGCCATCCATCAG SEQ ID NO:24 SEQ ID NO: 59 GCCTTCGGACCATGGTATCC SEQ ID NO:24

Example 5

Cloning of GFP-BLE Secretion-Induced Reporter Constructs

[0216] A fusion of GFP (Green Fluorescent Protein (Chalfie, M et al., Science (1994) 263(5148): 802-805) and phleomycin binding protein (BLE, present in pGBFIN-14 previously described in WO99/32617) was constructed by means of PCR. The GFP fragment was amplified by PCR using the primer set consisting of SEQ ID NO: 62 and SEQ ID NO: 63. The BLE fragment was amplified by PCR using the primer set consisting of SEQ ID NO: 64 and SEQ ID NO: 65 with pGBFIN-14 as template. The overlapping PCR fragments obtained were used in a fusion PCR with primers SEQ ID NO: 62 and SEQ ID NO: 65, resulting in PCR fusion product GFP-BLE (SEQ ID NO: 61). The GFP-BLE fusion was digested with restriction enzymes PacI and AscI and ligated in the PacI, AscI linearized cloning vector pGBFIN-2 (described in WO99/32617 and EP98/08577) thereby placing the GFP-BLE fusion under control of the glucoamylase promoter, the resulting vector pGBFINBLE-2 is depicted in FIG. 11.

[0217] Promoter sequences of the secretion-induced genes were isolated by PCR using genomic DNA from A. niger WT-1 as a template and primers as displayed in Table 4.

TABLE-US-00004 TABLE 4 Overview of primers used for amplification of corresponding promoters Primer Primer sequence Promoter PCR product SEQ ID NO:40 ACTTCATTAATTAAGTTGATTGAGGTAGAGATGAGTTTG P1, SEQ ID NO:20 SEQ ID NO:35 SEQ ID NO:41 ACTTCACTCGAGATAATGTAGGCGACAAAGTAGCC P1, SEQ ID NO:20 SEQ ID NO:35 SEQ ID NO:42 ACTTCATTAATTAATTTGACACTTAACGTGGTAAGG P2, SEQ ID NO:21 SEQ ID NO:36 SEQ ID NO:43 ACTTCACTCGAGTTCTCACAAGCAAATCCGAGA P2, SEQ ID NO:21 SEQ ID NO:36 SEQ ID NO:44 ATCCTACTCGAGTGGTGCCTGTGAACGAGGTCA P3, SEQ ID NO:22 SEQ ID NO:37 SEQ ID NO:45 ACTTCATTAATTAATGTGGATTTTGGATAGTAATTAAAG P3, SEQ ID NO:22 SEQ ID NO:37 SEQ ID NO:46 ATCCTATTAATTAAGATGGTTTGGTGTATAACAGAACA P4, SEQ ID NO:23 SEQ ID NO:38 SEQ ID NO:47 ACTTCACTCGAGGTCCACACTCTAATTGGGATGA P4, SEQ ID NO:23 SEQ ID NO:38 SEQ ID NO:48 ATCCTATTAATTAAGTTGCATCTGCGACAGACAGT P5, SEQ ID NO:24 SEQ ID NO:39 SEQ ID NO:49 ACTTCACTCGAGGCCTTTGGAGMTGTAATATCCC P5, SEQ ID NO:24 SEQ ID NO:39

[0218] The resulting PCR fragments (SEQ ID NO's: 35 to 39) were digested with restriction enzymes XhoI and PacI and ligated in the XhoI, PacI linearized cloning vector pGBFINBLE-2 depicted in FIG. 11, thereby removing PgpdA-amdS and replacing the glucoamylase promoter with the respective secretion-inducible promoter, Pind, resulting in pGBFINGFPBLE-1 to 5. pGBFINGFPBLE-1 is depicted in FIG. 12, and is representative for pGBFINGFPBLE-2 to 5. The fusion constructs of the respective inducible promoters with GFP-BLE was isolated by XhoI and FseI digestion of pGBFINGFPBLE-1 and subsequently ligated in the XhoI, FseI linearized A. niger expression vector pGBTOPSEL-1 depicted in FIG. 13. The resulting secretion reporter constructs, pGBTOPGFPBLE-1 to 5, contain a fusion of GFP-BLE under the control of respectively secretion-inducible promoter P1, P2, P3, P4, P5. In FIG. 14 pGBTOPGFPBLE-1 is depicted as representative for pGBTOPGFPBLE-1 to 5. These vectors were used to transform A. niger WT-1 in example 6 and 7.

Example 6

Overexpression of Extracellular Protein Confers Phleomycin Resistance to A. Niger Expressing a GFP-BLE Reporter Construct

[0219] Transformation of the secretion reporter constructs pGBTOPGFPBLE-1 to 5 to A. niger WT-PHY-2 overexpressing the secreted protein phytase was performed according to the procedure described in Materials and Methods. Transformants were selected on selective regeneration plates supplemented with various amounts of phleomycin (0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 microgram per ml) as selective agent. All 5 reporter constructs enabled direct selection of transformants on the phleomycin containing plates whereas the negative control, WT-PHY-2 wild-type, was not able to grow on any of the plates containing phleomycin. The secretion reporter constructs pGBTOPGFPBLE-1 to 5 enabled growth on respectively 50, 35, 50, 35, 25 microgram per ml phleomcyin. Thereby demonstrating that the secretion-inducible promoters of these constructs are functional and direct selection on phleomycin after transformation is possible.

Example 7

Selection of A. niger Clones Expressing Extracellular Proteins after Co-Transformation of a cDNA Library and the GFP-BLE Reporter Construct

[0220] Aspergillus niger WT-1 was co-transformed using the secretion reporter constructs pGBTOPGFPBLE-1 to 5 in combination with a defined A. niger cDNA library encoding for 50 extracellular proteins and 50 intracellular proteins. Co-transformation events were confirmed by colony PCR. Spores of co-transformants containing the secretion reporter construct in combination with an expression library construct were grown on selective plates supplemented with different amounts of phleomycin (0, 5, 25, 50, 100, 175 microgram per ml). An average increase in phleomycin resistance was demonstrated for co-transformants containing the secretion reporter construct in combination with a library construct encoding an extracellular protein compared to transformants with the secretion reporter construct in combination with a library construct encoding an intracellular protein. Graphics representing these results for secretion sensitive promoters P4 and P5 are shown in FIGS. 15 and 16. These results demonstrate that an increase in phleomycin resistance of at least up to 20-fold was achieved.

[0221] Diversity of extracellular proteins produced by the co-transformants which contained secretion reporter constructs comprising promoters P1 or P3 in combination with a library construct encoding an extracellular protein was demonstrated. Co-transformants were individually grown in microtiterplates and on day 6 the supernatant of these cultures was subjected to protein analysis by E-PAGE.TM. High-Throughput Pre-Cast Gel System (Invitrogen) and mass spectrometry (MALDI MS/MS).

[0222] The E-PAGE.TM. 96, 6% Gels (Invitrogen) contain 96 sample lanes in microtiterplate format and 8 marker lanes for protein separation. Supernatant and marker (E-PAGE.TM. SeeBlue.RTM. pre-stained standard (Invitrogen)) were loaded on the gel according to suppliers manual. After electrophoresis of the gel, SimplyBlue.TM. Safestain (Invitrogen) was used for staining according to suppliers manual. The scan of the E-PAGE.TM. gel was edited by the E-editor program provided by Invitrogen, which aligns the protein samples in microtiterplate format. Extracellular proteins produced by co-transformants with secretion sensitive promoters P1 and P3 are displayed in FIGS. 17 and 18, and are summarized in Table 5. These results demonstrate that up to 77% of the analysed phleomycin resistant colonies secreted proteins in amounts detectable by E-PAGE. Based on the size of the observed protein fragments on the gels, the diversity of the clones transformed with the defined library was estimated. These results demonstrate that a diversity of up to 35% could be observed based on E-PAGE analysis.

[0223] The mass spectrometry results are depicted in Table 5. Up to 58% of the analysed phleomycin resistant colonies secreted proteins in amounts detectable by mass spectrometry. Diversity was estimated based on the identity of the detected peptide sequences. These results demonstrate that a diversity of up to 100% could be observed using mass spectrometry.

TABLE-US-00005 TABLE 5 Summary of E-PAGE and mass spectrometry results. Transformants analysed Positive Diversity Assay Promoter No No % No % MALDI 1 26 14 54% 14 100% MALDI 3 31 18 58% 12 67% E-PAGE 1 26 20 77% 7 35% E-PAGE 3 31 20 65% 6 30% The assay used for analysis is depicted in the first column; the second column identifies the secretion sensitive promoter-reporter construct; the third column depicts the amount of transformants that were analysed by either assay; the fourth and fifth column depict the amount of colonies scored positive for secretion of a protein and the respective positive percentage; the sixth and seventh column depict the number of colonies secreting distinct proteins and the respective diversity percentage.

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 65 <210> SEQ ID NO 1 <211> LENGTH: 168 <212> TYPE: DNA <213> ORGNISM: Bacillus subtilis <400> SEQUENCE: 1 cgactcagtc ctttcatata caatatgaag tgtaccgttt tccgcacttt ttcacaattt 60 cccataatct tttcattttt atcccacagt ttttgtttat gataaactca agtcataaac 120 ctatcaatat aaatagacat gtgaaaatag agaaacggag tgaacatg 168 <210> SEQ ID NO 2 <211> LENGTH: 277 <212> TYPE: DNA <213> ORGANISM: Bacillus subtilis <400> SEQUENCE: 2 ggctcttcac atcctttcaa cgtcattata aactagtttt aacatacggc aggcaatttt 60 cataatttca catattcttt tcatttttat cccacaatgt ttgtttatga tatggttaag 120 ggaagaaagg aagagaaaaa gagcgaggaa gatgtaggat gatcaaacat attctatttt 180 ggtgctttgc ttttctcctc attattggga caattgaact tgtacatgcg atgaatatgt 240 aacgataaat gaataaccgt taaaggagtg taagaac 277 <210> SEQ ID NO 3 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 3 gtgagcggat aacaatttca cacagg 26 <210> SEQ ID NO 4 <211> LENGTH: 70 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 4 gtggctcagc ttttttaagg aagggaggct ctcacctccc ttccctttat ttgtagagct 60 catccatgcc 70 <210> SEQ ID NO 5 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 5 cgtgaggtac cggcttctgt ttctgcc 27 <210> SEQ ID NO 6 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 6 catcacgaag cttatccatc atgttcactc cg 32 <210> SEQ ID NO 7 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 7 gaccggtacc tcaggatctt tcgcc 25 <210> SEQ ID NO 8 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 8 ggccatcaag cttataatcc atgttcttac actcc 35 <210> SEQ ID NO 9 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 9 gaaagattgt ttcagaagc 19 <210> SEQ ID NO 10 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 10 acagcgttgg gatccaagcc cttccatttt ggacatttgg 40 <210> SEQ ID NO 11 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 11 gggcttggat cccaacgctg tcgacgttgt aaaacgacgg 40 <210> SEQ ID NO 12 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 12 cgcatagctt tccggtcgcc gcagctatga ccatgattac gc 42 <210> SEQ ID NO 13 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 13 gatgattcgt ctttttgtag tg 22 <210> SEQ ID NO 14 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 14 cgatcaacat atgtgttcac g 21 <210> SEQ ID NO 15 <211> LENGTH: 1050 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 15 ataatgtagg cgacaaagta gccgggcatg cgaccggagg actcggtcag aggcacgaat 60 atgagaggcc agaacgccgc acccatgttc caagatgttg tggcccagta aaggttagga 120 aatggggtgt tttgaacgtg gaatcgttct tccatccagt tgttgccgga gccgtaggaa 180 ccggccggga taccagtgag gatggaacta tacctattag cactgccatc ttcgagaaca 240 aggctaagac atacatgaaa caaacggtga ccgtgagaag ccatttataa gaggtgctcc 300 aattgaacct gaatatgtta aaactagtat actagcaact gcatgcttga cgtacggatt 360 gtccgggtca tccttgccgt tccaggtatg gatctcaagg tcctccctgt cacgcagatc 420 attgtggctg actaccccga catgggggtc ccggcgaaag aacgaccgca ccctgcaacg 480 tcacttagtg gctgtctgac agggattgat tggcaatgat tggcagacta accgtcccag 540 aaacgaatca tcgaactcat tggtgtgcac tgtgctagcg cggcgcagcg acgggcgtgg 600 ctcctcggca ggggtggaca tgactttctc tgtgtagatt ccttgcaagg tatctgcttg 660 acagatgcag agatgatgtt gagtaaaata gggtgcagag accctgcaga acctgcatcg 720 gccggcggcg acatgtcgtc atgaccgacc cgggccgagc ggaattaatg aaactcggaa 780 cagccagggg aatccgacgc tttgagtggc caatccatgt ctatccacat ctgcattgaa 840 gtggatgaaa ggtggaggag ggctggttgc caaattctgt tagggcttgg aatgtaagca 900 accggtttcg gtcatgacat catcacagcg atgacttcat cactctcgga cgagcatata 960 tagttgtgcc tgtcgtcgta tctcaacaaa catcaacaac aacaacaatc atctactgca 1020 tttaccaaac tcatctctac ctcaatcaac 1050 <210> SEQ ID NO 16 <211> LENGTH: 1032 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 16 ttctcacaag caaatccgag aaacgcaatt gaccatatgt ccttagtatg agggctagca 60 ggaatacttg tttgtcgatg cataccgact aataaaaatg gttcttcttt ttcgcgcagg 120 gatttggttt ttcatttttc atttttcata ttttatgtta ttttatttta tttttttgtt 180 ttattttatt ttattttttt tttttaaata agactgggtg ttttcgtctc ccacccctgc 240 tgaggaactg tttgatatac agaagcctga gatacttgag attatgtgac aaagaaattg 300 taaacctgaa tgtgactaag gcagaagaga gcaggtgagg ccacacaata ctgtacaccc 360 acttccgatc gtcatcgaaa tgagcgttgt gggtactgga acgacaaacc atagatcgga 420 tctgcatccg aataggcttt actttgtaca ttggtaggag tttgaatgtt tgaagatccg 480 gcagatgatc acctgctgca gggaataatt tgaaacctgt ccgtctgctt tgacccaatt 540 gacgaggtac gtgatttgat gctacccaag cagtatgaat gcgacggagg tggctatcct 600 taccttgtca tatgcaactc ataacgggta gtccccatgg agccacggct atttatttct 660 aagggccaac agtgaaagat tgtgagatcg tatacctcct gggtatgacg caaccagggt 720 gagcactggc attgctccaa tccatatggt ggaggtatgt ctccgagacc atgacgcatg 780 tagaccctga cctgtccctg atccagggat ctggtttgtc acttggcgta ttttaattaa 840 ctgcgggtca gggtcttgcc gtccagaccc aatggatgaa ttgcctgcgt ggccctgcag 900 ttccagcgat cggcacggca cggctatgac gtcggtgggt gttccgtttg atatttaacc 960 atagaagaca gccttgttat gctggttcct gccgcaaaat aaaggtctat ccttaccacg 1020 ttaagtgtca aa 1032 <210> SEQ ID NO 17 <211> LENGTH: 1035 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 17 tggtgcctgt gaacgaggtc accactgata aattgtctgg atccgtaacg tctgtatcag 60 tggatggtac tgaaaagttc caaagattga gggcagcata tgtagcacat ggtggtagct 120 ctaagtggtt cgaaacttcg atgaatgggc gtgatataca ggggggtagc acctgtatag 180 ttgatgtcag cgaaggcacg ggccatgcag tgatagacaa gaaaagaggg attgaataaa 240 agactcgatt ctgaagggga gtaatacatc tttcggcttc tcaccacccc agatgtaggc 300 gtgtgtaagg taagcaagca caacatatgc tctccgccac tcaggttccc ccttcaactt 360 cttcgtagag agtataccca ggtcatcgac agacctcctg atagtaccag actgaatcag 420 gccaggcaag tcctgagcga tttcctccca cggggaatag taagggtctt ccaatctccg 480 tacgggagga acttccggga ggaacccgtt ctggagggag acaccgtacg caacaagtga 540 aatgtcttgt gtataaagca tgataatata tgcacttcag tagacaactt gatattcaag 600 cctccttgaa atgatcctca tgcaggtaag agtagatcca agtgtttata ttgtattccc 660 aaggagggac ggaagaaaac cggatccaga gggctcgaga tagggccacc tgcatgagca 720 aagacgggtc atttcccgtt tcacaacgac cattctcagg cagaaaggca gtggattagc 780 ggaaaactta gcaaccatcc ggatttctgg agtggatgct ttgttatctc acttccggca 840 acacggatgg tgaggcggaa gcggaacggt atccggatcc ggagggaggg acaaaccgaa 900 gtgccttgtg tcacacgccg gtccatatat aaatgttaat tggtgacggt cagcggattg 960 tttctggtgt tcatccattt tttcttgata tcctggaccc agttattgca ctttaattac 1020 tatccaaaat ccaca 1035 <210> SEQ ID NO 18 <211> LENGTH: 1071 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 18 gtccacactc taattgggat gagcattgcc tttcaggaac ttgacttttt gtggttgcat 60 cttttgttgc cttacccacg caggcttcgt ttatgagata gcgatttcag gcctcaaatt 120 gctgtcatca cttccggccc caaactcgtg ggcggtgccc gcccatcgct ggcaagaagc 180 acagactaac gccttacttc gcctagttgc acacgatgca tcaagacttc ctcctatttc 240 acaacgacca gctccttgcc atttgttgtt tcgaatgccc ccttccccgt gccttgcatt 300 tctaattccc atctataccc ttttgcgctc tcacgcccgc ctgactatca atttttctgc 360 cagcataacc ctgcgtgcag agcggttgac agcaggcttg ctaagagtct tcgtctagaa 420 gaactgtctg ggtggattcc ttttcggatt aacttttgct attttaaaag ctgattgcac 480 tacggagtac acccgcaatc tgattctcac tgatactcca attaaaggcc actaattgct 540 gacatttgta acatatttgc gtttgctttc cggttgaaat tggcggcttt gtgctttcga 600 tcgctagcag tctctgtgca tgtacaactg atcggctatt cagtcctatt cggaagcggc 660 actgagaggc ggagacaagc cacagatacg acgagtctga ttttgagacg ggcgcatcca 720 acgaaagcct gcgattgcaa ttgaagatct caattggggg actgacaacc taatacctaa 780 acatatcgag gattaggcgc caacacgagc atgggaagct ttggtgcatg cgacggctga 840 tccatttcta acaaccgttt tgcgcgcgcg tccgaggtaa ggctcccgat cccttggcgc 900 tttgcatgtt tcactccgat gcatctactg tttatatgag tgcggtgggc aagctgtgcc 960 caccgactga gcattctcaa ccggcccaat gctgtgggat cgatcctccc ctccaaagtt 1020 gtcgctcggt tacacttgat cggagcttgt tctgttatac accaaaccat c 1071 <210> SEQ ID NO 19 <211> LENGTH: 1116 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 19 gcctttggag aatgtaatat cccttgatca ccccagactc acatccagat tctgactgac 60 ctcgtgcata gccatattta acttggtccc tgatgatcaa aacagtacat ggaactgggg 120 ccgaacattt ccctagccaa aggagcatca gggattcaac gtgatcagta cagccttgta 180 gctcgctgaa actgccctgg agccaagcca aagcctagac agcaaggtgc atgggcgagc 240 tttgcgagga tttagttgtt atttagtatg ccaagattaa gccacgcaga atggcttatg 300 gccatagtag caacaacggc cacggaacgt tcttccatgt ggtatgggga aagccgccgg 360 atatttgggg agggcgtgat tgttccggtg gctatctttc cggtggagat aacggcggtg 420 gaccatcaaa actcctcagc ccggattgct cggactccgc atcgtcaagt atcggtttca 480 tgacagccat tcgactactt agaagacaca aatagctatc caacttaccc ggacctttga 540 taataatttt cagcagatct gtcgatctca gagacaataa tgccaccgtt ctgtgtggaa 600 ggcactgcgc cgtggtttaa caataataaa actccaaatt gcccaaaagc tagagcgaag 660 gaaagtcgag aagcccagtc tagactggcg ggctgatcgt ccggttcaca acctgattga 720 caaatcatct gtgaccctcg atgttccgcg gcgttgagcg ggtgagaatg tggatcctag 780 tggggaagac ccaattgccg ccggaaaagg tgagcttgtg ctgcacatgt ccggtggctc 840 aattcagggc catgccagga cattgctctt caaagggatg cgattgctat tccctattta 900 cttctttaac ccgctgacgg gcctgtctgg accgagaatt cttggtcaag tttcagtgga 960 gcgatccagg aaagaacatc cgatatggac agttgccctc gatctcaggt ctgtagtcgt 1020 gtgacacaga ggcctctact cgcttgcctt gcatgtcgga gctcaaaggt gcgatgtgaa 1080 ttaacacgag gagcgactgt ctgtcgcaga tgcaac 1116 <210> SEQ ID NO 20 <211> LENGTH: 3191 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 20 ggagttacgt cccagagatg ctgcggacaa tgccgatgcg gcatacttct catacgcatc 60 cgtcaagtaa ttcacaacag ccatgtagat actgtagatg ccaataccgg tagccccgat 120 gccgagggtc gggacaatcc aaggcacaga aggaaaactg gcccaaccat accagaacag 180 cccgcctgcg aacagcaagc tgccaatgat actagtatac agacgggcct ccgggatggg 240 tttcccgggc ttctctttgt tgcgtttcgc cgaccgcaaa tacagcatat cctgaagagg 300 attgacgaga gtaccgatta gagcacccac ggagatggcc aattgaatgc acccagtttg 360 caaagtattc atgccgtagt tggtggagaa ggtctgaacg acactgctga agaagagata 420 caggataccc caggcgaaag agatccagag ggtgaagaag gcaacgacgg gctcggtgag 480 gagcattcgt gtgggccgtt cgaaggagat ctgcatcaga cgccagacgc tggtgttgtc 540 tagctcggct tcggcgtaga tggggcgctg cttttctttg cggagcttct tggcccgacg 600 ggtgaggatc acgtccgcgc gggtttcgta caggatgaac cagaagatgg gtaggagccc 660 tgttagatag ataatttgga tgtagaagat ccagcgccac ggggcggttt tgtggatggc 720 tacgatggcg gatccgatga atgggcccag ggcaattcca accacactgg taaaaccgaa 780 gagcgacata gggagactcc tcgctttgtc accataccag acatcactga tgctaccgcc 840 gacgatgttg atcgatacgg aggacgcacc accaccgaag aaacgcgtca ctacgagagt 900 agcgtagttt tgggcgaagg cggaggggaa tagcgagatg atgagaataa tgtaggcgac 960 aaagtagccg ggcatgcgac cggaggactc ggtcagaggc acgaatatga gaggccagaa 1020 cgccgcaccc atgttccaag atgttgtggc ccagtaaagg ttaggaaatg gggtgttttg 1080 aacgtggaat cgttcttcca tccagttgtt gccggagccg taggaaccgg ccgggatacc 1140 agtgaggatg gaactatacc tattagcact gccatcttcg agaacaaggc taagacatac 1200 atgaaacaaa cggtgaccgt gagaagccat ttataagagg tgctccaatt gaacctgaat 1260 atgttaaaac tagtatacta gcaactgcat gcttgacgta cggattgtcc gggtcatcct 1320 tgccgttcca ggtatggatc tcaaggtcct ccctgtcacg cagatcattg tggctgacta 1380 ccccgacatg ggggtcccgg cgaaagaacg accgcaccct gcaacgtcac ttagtggctg 1440 tctgacaggg attgattggc aatgattggc agactaaccg tcccagaaac gaatcatcga 1500 actcattggt gtgcactgtg ctagcgcggc gcagcgacgg gcgtggctcc tcggcagggg 1560 tggacatgac tttctctgtg tagattcctt gcaaggtatc tgcttgacag atgcagagat 1620 gatgttgagt aaaatagggt gcagagaccc tgcagaacct gcatcggccg gcggcgacat 1680 gtcgtcatga ccgacccggg ccgagcggaa ttaatgaaac tcggaacagc caggggaatc 1740 cgacgctttg agtggccaat ccatgtctat ccacatctgc attgaagtgg atgaaaggtg 1800 gaggagggct ggttgccaaa ttctgttagg gcttggaatg taagcaaccg gtttcggtca 1860 tgacatcatc acagcgatga cttcatcact ctcggacgag catatatagt tgtgcctgtc 1920 gtcgtatctc aacaaacatc aacaacaaca acaatcatct actgcattta ccaaactcat 1980 ctctacctca atcaacatgc ctatctccat tccctccgcc tccagcgtcc acgacctctt 2040 cagcctgaag ggcaaggtcg tcgttatcac cggcgcttcc ggccctcgcg gcatgggcat 2100 tgaagccgcg cgtggctgcg ccgaaatggg cgccaacatc gccctcacct actcctctcg 2160 tcctcagggt ggtgagaaga acgccgaaga acttcgcaac acctacggcg tcaaggccaa 2220 ggcctaccag tgcaacgtgg gcgactggaa cagcgtcaag aagctcgtgg acgacgtgct 2280 ggccgagttt ggccagattg acgccttcat tgccaacgct ggcaagacag ccagcagtgg 2340 tatcctggat ggttccgttg aggactggga agaggtcatc cagacagacc tgacgggtac 2400 cttccactgc gccaaggcgg tgggcccgca cttcaagcag cgcggaacgg gcagcttcat 2460 catcacctcc agcatgtcgg gtcacattgc caacttcccg caggagcaga cgtcgtacaa 2520 cgttgccaag gcaggctgca tccatatggc ccggtcattg gccaacgagt ggagggactt 2580 tgcccgcgtg aacagcatct ccccgggtta catcgacacg gggctgtcgg actttgtgga 2640 taagaagact caggatctgt ggatgtcgat gatccccatg ggacgcaacg gtgatgccaa 2700 ggagctgaag ggtgcatatg tctatctcgc cagtgatgcc agcacataca cgacgggtgc 2760 cgatttggtc attgacggag gatacaccgt gcggtaaatt atcatccgcg gtcggataga 2820 aaataatgtt tatgacatgt atgtaatgaa tatgaatgtt caacaatagt cttcaattct 2880 tcacttgcta gtatcctgag tatcttgtcg tatagctcaa caacctggca tggccgggcg 2940 cgcagtgtag ctctcccaag tctatggata caacttgcgt cagtgtggat ctttggctca 3000 gattcggagg ggcagtgggg taaacaagaa cacgcagcca ctctcggctt tgcggatccc 3060 cacggaacgc cagtcaaccc tgaagattca gtccgggcct gggctcggac tgatttggtg 3120 aggggattct gcatccgtcc aaaccttatc atcgttggtg agacggactc cggacagcca 3180 ggagccttat t 3191 <210> SEQ ID NO 21 <211> LENGTH: 4031 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 21 ggggtcaaag gacacaatgt cgaaatagcg agattgctgt gcatcatcgg ggctgtctac 60 agtccgctgg atttctttcc ctcggttaag tatttcctgc acgccggacc cgccagggcc 120 acctgtattc ctagtaagcg accgtggtgg ggtgacagaa tggtggcggt ggtggcattg 180 ctgcctatac ctgggttcac gattatggag ccaccgtaac gctcgtcggc aggatctacc 240 tgtgcaggca gttttatgac ggccagggca gccgttttat tttcaatggc actagctgac 300 cagtctaggg gaacttcgag acgtgcacat agatagtcct ggtagcaggg gatgaaattt 360 agtgctgagc ctggcgcagt ctggtaggag cgggttagac tgtgagaaat tataataggg 420 cggatgagat cagtacgcta ctccaggaga agtccccggc ttgcaccgca gacggcatca 480 tgggttggtg tcgcagggag gagggtgagt tgagtaagag agaattaatg aaaaggatgg 540 agtagccgac catataagta ctggatgaag tgagcgagtt agcgtcagcc acgacgggct 600 gacatgcatg atcggggcac cgccggcaac cgatacagcc tcagctcgac gggaaaacgt 660 ggttacctgg acttagaaga tgataataca atgaaaagat agcgtagatt gccataaata 720 cgccacaatc tatttattag gcaacaatca agtaagagca gatttggcga ctcatagtag 780 tccccatgga ggggcgcgat ggttttcttc gccgaaataa tcgcttaatg cctctaacga 840 agatctcaga acctccaaac tgtagtattc ttctcacaag caaatccgag aaacgcaatt 900 gaccatatgt ccttagtatg agggctagca ggaatacttg tttgtcgatg cataccgact 960 aataaaaatg gttcttcttt ttcgcgcagg gatttggttt ttcatttttc atttttcata 1020 ttttatgtta ttttatttta tttttttgtt ttattttatt ttattttttt tttttaaata 1080 agactgggtg ttttcgtctc ccacccctgc tgaggaactg tttgatatac agaagcctga 1140 gatacttgag attatgtgac aaagaaattg taaacctgaa tgtgactaag gcagaagaga 1200 gcaggtgagg ccacacaata ctgtacaccc acttccgatc gtcatcgaaa tgagcgttgt 1260 gggtactgga acgacaaacc atagatcgga tctgcatccg aataggcttt actttgtaca 1320 ttggtaggag tttgaatgtt tgaagatccg gcagatgatc acctgctgca gggaataatt 1380 tgaaacctgt ccgtctgctt tgacccaatt gacgaggtac gtgatttgat gctacccaag 1440 cagtatgaat gcgacggagg tggctatcct taccttgtca tatgcaactc ataacgggta 1500 gtccccatgg agccacggct atttatttct aagggccaac agtgaaagat tgtgagatcg 1560 tatacctcct gggtatgacg caaccagggt gagcactggc attgctccaa tccatatggt 1620 ggaggtatgt ctccgagacc atgacgcatg tagaccctga cctgtccctg atccagggat 1680 ctggtttgtc acttggcgta ttttaattaa ctgcgggtca gggtcttgcc gtccagaccc 1740 aatggatgaa ttgcctgcgt ggccctgcag ttccagcgat cggcacggca cggctatgac 1800 gtcggtgggt gttccgtttg atatttaacc atagaagaca gccttgttat gctggttcct 1860 gccgcaaaat aaaggtctat ccttaccacg ttaagtgtca aaatgagtgg ggtaagcttt 1920 gcccgtcagg ccttactcca ggctgctctt ctttcgattg tctccgcaat cccggcgccc 1980 acggccttcg tcaacaatgt cgctccgccc gagcccatca tcaccccttc gccggttcaa 2040 catcgacctt ctcgagtcgc tggtcgaaac attctgagtg acgtcgattc tgatattaac 2100 agcatccttt ctggccttgg atccgaccta ccctcctggg tcgcatcggg tgtgcccaac 2160 tacttccagg gtttccctac tggggatgca gtggtgagct ccttgggctt gaacagcgct 2220 gagttggcag ccttgcccac caatgtcttg aatatcgacc catatgccaa ctggactagc 2280 tctggctgga acgttcgctt ccacggaaac gtctacaagc agcccaacac ctccatctcc 2340 gacctcaatg atttggctga tgttttcctc ggcaatacaa gcatctccga cctttccgaa 2400 tccgagcaga aacaggctcg gaatttaaca gcggaaatat tagtggtcca gcaagctcac 2460 gtcgccgtaa acacaatcca cctggagcct gcaccgagtc agggatccag cggacagtca 2520 ggcggcggtg gatcctctaa taccactggc ggcacccaag acctcactct gccctacaac 2580 actacagttg aaggcgattt tgacactttc gtaccgatta gcagcaatgg cttgacggca 2640 ggtaatgaaa cttcggcgat acaacggctt aacgtccatg tggagggcgc gacaatagga 2700 aacagtactg cctatctcgt acctccgacc gggctcactg tcgtttcgga catcgatgat 2760 atcctacgcg tcaccaaaat ctatgaacca gagcaaggtt tactcaactc atttgcgcgc 2820 cccttcaggc cgtgggagaa tatgccggac atctatcgca actggtcgat cagtctcccc 2880 aacctgcact ttcactacct agtaagtctg ctagatgtcc ctgattgcat tgttgctgca 2940 tactcaccac tacactacag acgacgaccc ccgaacaagt caccaggaac tacatgcaat 3000 tcatctacga caactacccc ggtggatctt tcgacacccg tcccctcaac tttagcgacg 3060 tttccgccac cttgtcgatc cggaaattcc tcctgcaaaa ggtcttcgag acctttccgc 3120 agcgcaagtt catcctcatc gccgacacca gcaacagcga cgtcatgcgt gactatcctg 3180 aaatggcgac cgacttccct ggtcaagtgc aatgtatttt tctccggaac accagcgcca 3240 ccgactcagg ggacaaattc ccatacgata cctctgggtt taagaagctc aatcagtcaa 3300 actacatgtt cttcttgcat cccgatgact tgacgaatct agacatcgcg aatggccagt 3360 gttacaacac atccattccg cagaatttaa cgtttagtta tcagggtttg ccattggggc 3420 tgggtgatga acccacagct gtcaatgggt ctgccaatca tactgctgag tcggcggccg 3480 gtttgtcggt aaagggtggg actgatatgc agggtttgat ctttctgttc accctggtta 3540 cggcctattc cattttcttg taaatatcag ttacgatgta tgagagataa cgactttgat 3600 atactcacgg ctcaatttac gttccaaaac cgaatacagc ggatatctat gctcgatgca 3660 aggactacca ctatgttaca ctactaaggg tttatctact atgggaaata ttagtgcttg 3720 gtttgttaca gctcactcga gcttgcgtta ctaaccactt ttatcggtcc atggatccac 3780 cgcccaacgc agttatccca ggaggtggcc gcaatggcca gtcaagtcaa ttaaatctcc 3840 tctgctagga attgcatggg ttttgacaga cagaactcgg ggaaagtatc gatattcaat 3900 gatctgacgg tggctccgaa attcgctagg ctagcccttc atcactagta atattagttg 3960 tccaactttc gcgctttaca ctttccatag caaaccccca attcccagcc aaatcctcat 4020 atcctctagg a 4031 <210> SEQ ID NO 22 <211> LENGTH: 3771 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 22 gcttctatgg tatcaagggt cgcgtagcca aaagtttaac tgcatcgctg cggtattagt 60 cggtgtagct ggctgcagct ttcgttgtgt ccctggtctg tttaagaaac ggtatcagat 120 ctgtcccgcc cgtccccctg acaccggcat tgagtttctc atgagaattg tccactattg 180 tcgacgttgt tgtggctagg tttgttctcg aagatacttg gtttggcggc ggagtccttg 240 cagccatgat gatgtatcga gtcaccatcc gcacatgctt atcgcgaaat cccccaagtg 300 acataaccgc catgttatat gcatccctca aagcagatcc gggcttgtga cttaatacat 360 aggatctcac attagacgtg cggagcatca tttccaagaa gcgtcggtga gggcctggca 420 tgtagtttcg catgttcttg aatatacagt cagtatcgac attgctttgt ctggataact 480 cctcgaagat cagtccttac ctgcataaaa ccagtcatga cttgaggatt cgacttgccg 540 tgaggcttgg tctccccagt ggccgaatgt tccacaccca agaaaatgtc gaaggcttgg 600 atcagagaac tttgagcatt gctaccacca ctgtattgat gccattcgcc tcgtccttcc 660 ccaacatcat agaatacgcc gtttggcagc ccggccgatg ccatgttctt gctgccggct 720 aggtatggac gtaattgatg gtaaaagacg ggcggcgcac acttttcata catgcgttcc 780 aagaggtcac cgagttcctt gagaccataa ttgagctgct ccagacgaga aataacacgc 840 tggctatcat caacattagc tgcatgcata gcatccagca ttagctcgat gagctgtgct 900 cccttggcct cgatagcgac ggagaggacg aaaaaccatt cttcgtcttt ggtgcctgtg 960 aacgaggtca ccactgataa attgtctgga tccgtaacgt ctgtatcagt ggatggtact 1020 gaaaagttcc aaagattgag ggcagcatat gtagcacatg gtggtagctc taagtggttc 1080 gaaacttcga tgaatgggcg tgatatacag gggggtagca cctgtatagt tgatgtcagc 1140 gaaggcacgg gccatgcagt gatagacaag aaaagaggga ttgaataaaa gactcgattc 1200 tgaaggggag taatacatct ttcggcttct caccacccca gatgtaggcg tgtgtaaggt 1260 aagcaagcac aacatatgct ctccgccact caggttcccc cttcaacttc ttcgtagaga 1320 gtatacccag gtcatcgaca gacctcctga tagtaccaga ctgaatcagg ccaggcaagt 1380 cctgagcgat ttcctcccac ggggaatagt aagggtcttc caatctccgt acgggaggaa 1440 cttccgggag gaacccgttc tggagggaga caccgtacgc aacaagtgaa atgtcttgtg 1500 tataaagcat gataatatat gcacttcagt agacaacttg atattcaagc ctccttgaaa 1560 tgatcctcat gcaggtaaga gtagatccaa gtgtttatat tgtattccca aggagggacg 1620 gaagaaaacc ggatccagag ggctcgagat agggccacct gcatgagcaa agacgggtca 1680 tttcccgttt cacaacgacc attctcaggc agaaaggcag tggattagcg gaaaacttag 1740 caaccatccg gatttctgga gtggatgctt tgttatctca cttccggcaa cacggatggt 1800 gaggcggaag cggaacggta tccggatccg gagggaggga caaaccgaag tgccttgtgt 1860 cacacgccgg tccatatata aatgttaatt ggtgacggtc agcggattgt ttctggtgtt 1920 catccatttt ttcttgatat cctggaccca gttattgcac tttaattact atccaaaatc 1980 cacaatgtcg aagtcgaatg gcgtcagcct ggcattcccc gccgaagcag cgacaaagga 2040 atatgcagca tctctagact cttctgatag acttgctgca tttcgggaga agttcatcgt 2100 cccatcgaaa gcaaatattg cttccaaaaa gctagcaaag cctggtgggt tccatcctgg 2160 caactgtaga cctcgtcact aatgtcacag gcctttcgcc cgagtcatgc atttacttct 2220 gcggtaactc acttggcatt caacccaagg ccacagcaaa gtacttagaa gcacagttgg 2280 acacttggtc atctattgga gttggcggcc acttcactga tcttgagggc tcacccttga 2340 agcaatggca gctgctctca gagcaagcag ctgattcaat gagcaagatt gtgggagcaa 2400 agccagaaga agtcgcagcg atgggaacac ttacaacgaa ccttcatctt ctgctggcta 2460 gtttctataa accgactcaa acaaagcaca agatcttgat ggattggaag gctttcccaa 2520 gtgatcacgt aagttcgaaa gaagctgcct tctgctatca cggcactgac gattatatag 2580 tacgctatcg aatctcatat tgcctggcat gacctggatc ctaaggagtc tatggtgctc 2640 atcggtccag atgaaggcga atacgagata tccacccaga agattttctc ttatatcgac 2700 aagcatgctg atgaggctgc tatgattctt cttcctggta ttcaatatta tactgggcaa 2760 ctgttcgata tccagaaaat tacaaaatat gcccattcgc gcaacatggt tgttggctgg 2820 gatctggccc atgcgttcgc taatgttgag ctaaagctgc atgattggaa cgttgatttt 2880 gcagcatggt gcacatataa gtacggaaac gccggtcctg gagcaatggg ggggcttttc 2940 gtgcatgaac aacacggaga ggtcgattac agcgcgggag aagatgcacc caaattccgc 3000 catcgtctga ccgggtggta tggtggcgat cgatcggtaa gattcaagat ggacaacagt 3060 atgtgtgcac ttggctatag aacaattttg tgggctagta agctgatacg attgataacc 3120 agagttcaaa cctattcctg gggcaggagg atttcagata tcaaatcctt cggccatcga 3180 ccttgcgtgt ctttgtgccg ctttatcggt gtttgatgag acgtcaatgg cagatcttcg 3240 cagaaaatca ttgaagctaa cagcatacct tgagttcctt ttgctcaggg attatgaaga 3300 agaatctagg ccattcagca ttatcacgcc caaggacccc gaggcgagag gcgcacaact 3360 gagtctattg ctcaagcctg gtctattaca gaatgttgca cagaagttgc aagaagcagg 3420 aatagtctgc gataaacggg agccgggtgt tgtgcgtgtc gctccggttc ctttgtacaa 3480 cagcttcagc gaggtttgga catttgtgaa gatcttcaag gatgcactgc agcagtgatg 3540 ggtctcacta gatcatttgg tcaggactga gaccagattt gtcaagttcg ctccgatgaa 3600 tgatttttta gatagatacc aaattatttg attcttagat ttgttgaagc tctcactgcg 3660 acatggtcca cagccactgt gcaaaagcaa ataactgaag taggaactct atgatagacc 3720 cgtactgttg aggttataga tttccaaagc cgttccttgg atcaccactt c 3771 <210> SEQ ID NO 23 <211> LENGTH: 3562 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 23 cctgtgcttg tggtatgaca gagccactga tatcatctcc tctcatctcc agttgtaatc 60 ggaattgatc gagggggcca atcttccaca cagccccatg gaagacgcac agatgctttc 120 gatgccacgc tggcagacgg ccgccaactg tcatcgtcca gtgctgccgc cattcccatg 180 caaaagccaa cgctaagaag cgcgcgggcg ccgagtttcc acgcttgcag cttagttcag 240 cgacccagct cagtggcggc tcgctcatgc atagaaagat gggtttggac ccgcgctggg 300 cctttcatct gcaacactgc tcgcagacca tgtgtgactt ttggctgcaa aaaaaaagag 360 gctggcgaca gctgatccac tgtgctattc ctatctcatc ctccccggct ggatcgaaag 420 ctgcacggca ggggaaatca acagttgggc tctgacacgt atcgagctaa ctgatgcgct 480 gtccttccgc ctaatgccta ctaagtctta gggacgggac catcatccaa catgccacct 540 tgtccttcgt aattcctttt tgatctcggt gatggtggcc acccaggaaa tgacgttcct 600 caccttggct gcaattggtg cccgatgtct catctccgct cagaacggcg gcgagggcag 660 aataaaaatt gacgggcaca gcaggaacta atcaaacata ccggcggtct tctaaatgat 720 cgtatctccc ggaatactcc ctctttctac agaagcgggg atccattcgc cctatatgta 780 tagtggagct ttgccgcact gattcgcacg gcattatacc tccgcactgc cgtgccttca 840 tccacttgat tgtggccagg gaaatatcaa cggggcagat aacttgccct tgcatcatta 900 ttgctccgca cattcattca ttatcgctct gatcccacat cctcacgcag ctgcgaccgg 960 agtgtccaca ctctaattgg gatgagcatt gcctttcagg aacttgactt tttgtggttg 1020 catcttttgt tgccttaccc acgcaggctt cgtttatgag atagcgattt caggcctcaa 1080 attgctgtca tcacttccgg ccccaaactc gtgggcggtg cccgcccatc gctggcaaga 1140 agcacagact aacgccttac ttcgcctagt tgcacacgat gcatcaagac ttcctcctat 1200 ttcacaacga ccagctcctt gccatttgtt gtttcgaatg cccccttccc cgtgccttgc 1260 atttctaatt cccatctata cccttttgcg ctctcacgcc cgcctgacta tcaatttttc 1320 tgccagcata accctgcgtg cagagcggtt gacagcaggc ttgctaagag tcttcgtcta 1380 gaagaactgt ctgggtggat tccttttcgg attaactttt gctattttaa aagctgattg 1440 cactacggag tacacccgca atctgattct cactgatact ccaattaaag gccactaatt 1500 gctgacattt gtaacatatt tgcgtttgct ttccggttga aattggcggc tttgtgcttt 1560 cgatcgctag cagtctctgt gcatgtacaa ctgatcggct attcagtcct attcggaagc 1620 ggcactgaga ggcggagaca agccacagat acgacgagtc tgattttgag acgggcgcat 1680 ccaacgaaag cctgcgattg caattgaaga tctcaattgg gggactgaca acctaatacc 1740 taaacatatc gaggattagg cgccaacacg agcatgggaa gctttggtgc atgcgacggc 1800 tgatccattt ctaacaaccg ttttgcgcgc gcgtccgagg taaggctccc gatcccttgg 1860 cgctttgcat gtttcactcc gatgcatcta ctgtttatat gagtgcggtg ggcaagctgt 1920 gcccaccgac tgagcattct caaccggccc aatgctgtgg gatcgatcct cccctccaaa 1980 gttgtcgctc ggttacactt gatcggagct tgttctgtta tacaccaaac catcatggat 2040 gtgactaatt tggctgtcga ttgtcccgtt atagagaccg gttcccttgc tttgtggatg 2100 gcttcctccg aagtggacca gaaggccttg ggcaactttg cggccataac gggcctcgag 2160 aacccgttcc tgtccgcgat gctgtacaag attctggggt tgtctgttat gttatcgaag 2220 aaactcctcc gtgcacgacg attgcggcgc ctagacccca cccgagagac taaatcgctt 2280 catctttatt accatatcat ctggctttcg cgtgagggtc ttctgatatt agaggagttc 2340 gtcctgccac tggttgaggg atttatggaa ctcaaaattt tagcttacaa gctgcgcgcc 2400 tccttttacc atatattcgt cctatttcag aaccagcctg ctgtccactc gccgggtatc 2460 gttagtctgc cgagtagtac acctttatcc aacggcgtga cagaagcgga gtcaccatcc 2520 aaggattcca atttgagatt ttcatttcag ctgaagcccg acacgataac ggtttccggg 2580 aagcccagtt ccgcctcgga tagtgcgcca cgaggcaagg ttacgcaggc acccccgggc 2640 tttgcgccgg ttcaaccgcc caagtcgaac tcggcgtttc tcctccctgc tctcgattat 2700 acccctacag ctaccgcatg cttcaatcat gccgccctct tggcagatca attcctcccg 2760 gggtcgcacc cgctccgtct gtccatcaag cttgaatttg ccgcctacct ctacgattgc 2820 ctacatgacg ctcatgcctg tcgacggttg gccaagcaag ccattgccga tgtgtacaat 2880 gcgcaagagg gcatggacga cgagagcttc gaggatgcag ctgagattgt gggtattttg 2940 ggcaagatgg tgaagcgggg aaggaacaca agcagtgctg gaaatagcac cacggctgtg 3000 aaaacgcccc aggaggagcg aagtgagggc agtcgaacac catcctctca gacaacgttg 3060 aaaaggccgg cgcgagtgcc gaaaagcacc tcctcaccgg cgcgcgcgac caagccgacg 3120 actagcgagg ggatgtcccc cgctgtgccc gaccccacca tgatgaatcc catatgatgc 3180 ctttccctac gacctgtata tgcttcttct tggaactatg gagtcgcgca tgagcgcttg 3240 gcctgtgact tctacttgac ttgcacctgt ctgtatcatc tcggtaacgg agttgcggcg 3300 atagactgga attgggcatc agcttgcggg gagctgtgga atgagcgatt aagcgcgaca 3360 aaaacttgag gtggaaggga cagaccagcc aatttgtgat ggtgactgta gatagataga 3420 tgtgtgcaat acaaagtatg ttgttgatgt catgcacccc acttgtgtat gcttgtctgg 3480 acaatggacc tacacttagt cgcaacaact gtatgtctca agccaatggc catggggcag 3540 gatggatcga tggatctctt gt 3562 <210> SEQ ID NO 24 <211> LENGTH: 4361 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 24 cgacggacta acgtcgcgcg ccccatttca cgtcttgaag acagatgtgt atgcgattgc 60 tcgcgaaatt ctcatccgat tccgtcggct tgacctgaca tctggaatag acgaacagga 120 cttggaagct atcattcgta ttaccgaaga gtccaacgta cttctcactg actggtggaa 180 gtccgtcccg aaactgtatg acttcgatta ctggaaagca gacggtcgat gggaaatgat 240 tgaaggccag ctgcaaaggc ttccctccga agcaaggcaa caggtggaga caatatatct 300 acaggctgct gttctccagt tgacgtacga cggcatggtc atccaagcaa atagaccact 360 gttggagcga agaatcaaca ggctaacatc ttcgcgggcg atcgtggatg cgatgcatag 420 ggcgcttgac cgagccactg ctgctgctct ccgcatatcc cgtgttccag ttcacaagct 480 gaaaaatcat tttgctatag cagtcgtatc gatgcaagag ttcactgctg gtgtgattct 540 ctgtatacct cccacggtga agcctttaac ggccaccgct catgaggcga aggacggggt 600 ggtaagaata atacgcgcca gtcgaagtct caagagccac gatcgaatag caaggcaaac 660 tgaacaactt ctgacggaac tactcaaagt taccacccaa cgcgagataa cgcacgcctt 720 ggccgataat ctcgacacca atttcccctc cgatacgcgg ctaacaggct ccgttgttgc 780 cggcgcctca agtgactcct tgggtgcagg acgctccagg ccagttcccg gatccgcagc 840 gcaggcagtt gtagatccta tccaaggagc ctgggactca gaattcaatg ccctccctgc 900 cgtaacggga ccttatgatt tcctccctgc tcaagccttt caacagctgg acgatacatt 960 tggtgccttt ggagaatgta atatcccttg atcaccccag actcacatcc agattctgac 1020 tgacctcgtg catagccata tttaacttgg tccctgatga tcaaaacagt acatggaact 1080 ggggccgaac atttccctag ccaaaggagc atcagggatt caacgtgatc agtacagcct 1140 tgtagctcgc tgaaactgcc ctggagccaa gccaaagcct agacagcaag gtgcatgggc 1200 gagctttgcg aggatttagt tgttatttag tatgccaaga ttaagccacg cagaatggct 1260 tatggccata gtagcaacaa cggccacgga acgttcttcc atgtggtatg gggaaagccg 1320 ccggatattt ggggagggcg tgattgttcc ggtggctatc tttccggtgg agataacggc 1380 ggtggaccat caaaactcct cagcccggat tgctcggact ccgcatcgtc aagtatcggt 1440 ttcatgacag ccattcgact acttagaaga cacaaatagc tatccaactt acccggacct 1500 ttgataataa ttttcagcag atctgtcgat ctcagagaca ataatgccac cgttctgtgt 1560 ggaaggcact gcgccgtggt ttaacaataa taaaactcca aattgcccaa aagctagagc 1620 gaaggaaagt cgagaagccc agtctagact ggcgggctga tcgtccggtt cacaacctga 1680 ttgacaaatc atctgtgacc ctcgatgttc cgcggcgttg agcgggtgag aatgtggatc 1740 ctagtgggga agacccaatt gccgccggaa aaggtgagct tgtgctgcac atgtccggtg 1800 gctcaattca gggccatgcc aggacattgc tcttcaaagg gatgcgattg ctattcccta 1860 tttacttctt taacccgctg acgggcctgt ctggaccgag aattcttggt caagtttcag 1920 tggagcgatc caggaaagaa catccgatat ggacagttgc cctcgatctc aggtctgtag 1980 tcgtgtgaca cagaggcctc tactcgcttg ccttgcatgt cggagctcaa aggtgcgatg 2040 tgaattaaca cgaggagcga ctgtctgtcg cagatgcaac atgagaaatt tggagtgtgt 2100 ctacacaaaa tcacgaaggt atccctcgtc tatctttccg tgtgtattat tagccgagag 2160 tcacgcaatg ctctttgtat acaatgaaat aatgttggag ctagagaatg atttctgata 2220 gttatatgtt acaggacact caggaggtcg tcaactagtc cgaacacggc gtcctctcct 2280 gacactaatg cctcatcttc gccatgctcg gactctagtg ggacccatgc tggcatttcc 2340 attccccact cagatagtcc cgccagtgtg gacgaatgtg ggaacaacgg ccttttcgac 2400 gggggtaggt aatatgtcat gcttacctgc gacactcact caccattctt ctagaatctc 2460 taactcaatt attggtctct gggagcaaag ccatatcagg ccaaccacat atcgccatta 2520 agccacatgt tctgtctacc tatccccttg acaacctcct ctcagacatt gagggactcc 2580 tcgagcagcc agtctctcat ttggctcaaa gcaaggaaga tctaacaaaa ccggaccgga 2640 tgtttcgaac tcgccttcca gcggtctcag gtgaggacaa tgagtatctg ctaaggaaag 2700 gagctctgct gctcttgcca ccagccttgc gcaacgaact attggcggca tatgtcaact 2760 acgtccatcc gctattaccg attcttgatc ttggagactt tctctcacaa atcattgttg 2820 gagatgatgc tcaattcaag tcaccactat tataccaagc tgtcatgttc gcgggcagta 2880 tttttgctca gcgagacggc acagacgaac ctgaacagct aacgagggct ttatttgaac 2940 gtacaaaggt tagtaatggt tacaattaga catatcttag atatactgag cgatttccag 3000 gttctatacg agtttgaatc cgaatcatgt gcctacactc aaattcaagc tctcttattg 3060 atgactctct ggcatggaga tatgtcacgc cacaaaggcc cgtcttactg gcttgacgtc 3120 gccttctcca ctgcggaaag aattggccta ctccatgatg aagactggcc atgcgatagc 3180 cgttccttcc gatccgctat gtggtgttgc ctttacgtac gtgatcgaac aatctcgctt 3240 gggtctcgtc ggcctccacg gatgccagtc aacaaatatc cggactcaat tctccatctt 3300 atgagcgacg cacctcggga atatgacgaa ataattcttg agagtctagg tagcgcttct 3360 ctgatgctta caagcactta ccaagaacaa tcgaccatgc tcttcaagga acttgtcaaa 3420 ctctcctact gcgtcggggc aatcttagat tatctatacg aaggggcctg ggtccgaata 3480 ccaacacgtc gcagcagctt ttattcactc gcccctaaat gcagcatctc gccatccatc 3540 agaccaagct gtgagaaact actttactct tggattcaat tcctccctct caacgcagtc 3600 taccatccgc cacagttgcc cgccgatgcg gaaggggagt cagccgaaac agtcttcctt 3660 gtccacaaag ccttccttta cttgctgtac ctagcctcca tggcagtcat ataccgcaca 3720 ggcactcata gccaacaaac cagcacggca actccgaaca tggaaggtct acgggcatcg 3780 acgtcacaaa taaaaaaggt ccttgcggag ctgcagggca tgggactgct tcagttcctt 3840 ccaggggcat cagtcactat cctgatgttc gctgtggagg ctagtctgtt ggatctccag 3900 ggctcggata ccatggtccg aaggcaggct atgtcaaatc tatatgcctg tgaggaggct 3960 gcgttacact tgatgcaggc ttatcctacg gctgagatgg cggtattgaa gactaggaca 4020 gcccgtgcaa accttcttgg gttggttgag acatgaagac caatcatgga agctctgctt 4080 ccatcaaagc attgtgtctt tgcttaatga atgaatggat tatgagcgtt tcgcaacctt 4140 ctttaatgca gctacataat agagcataaa cagaggcccg gaaatatata atttggctaa 4200 gaacccggtt gcaagcaggc aagtcacgag gtgcagaaca taccatcaag tttatagatt 4260 attgtagggg atatcctcaa caatatctta cttccaccat tacgtactga agatcgaggt 4320 tatcggggac catgctgtcg tgtcaatagg tacgactcgt t 4361 <210> SEQ ID NO 25 <211> LENGTH: 801 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(801) <400> SEQUENCE: 25 atg cct atc tcc att ccc tcc gcc tcc agc gtc cac gac ctc ttc agc 48 Met Pro Ile Ser Ile Pro Ser Ala Ser Ser Val His Asp Leu Phe Ser 1 5 10 15 ctg aag ggc aag gtc gtc gtt atc acc ggc gct tcc ggc cct cgc ggc 96 Leu Lys Gly Lys Val Val Val Ile Thr Gly Ala Ser Gly Pro Arg Gly 20 25 30 atg ggc att gaa gcc gcg cgt ggc tgc gcc gaa atg ggc gcc aac atc 144 Met Gly Ile Glu Ala Ala Arg Gly Cys Ala Glu Met Gly Ala Asn Ile 35 40 45 gcc ctc acc tac tcc tct cgt cct cag ggt ggt gag aag aac gcc gaa 192 Ala Leu Thr Tyr Ser Ser Arg Pro Gln Gly Gly Glu Lys Asn Ala Glu 50 55 60 gaa ctt cgc aac acc tac ggc gtc aag gcc aag gcc tac cag tgc aac 240 Glu Leu Arg Asn Thr Tyr Gly Val Lys Ala Lys Ala Tyr Gln Cys Asn 65 70 75 80 gtg ggc gac tgg aac agc gtc aag aag ctc gtg gac gac gtg ctg gcc 288 Val Gly Asp Trp Asn Ser Val Lys Lys Leu Val Asp Asp Val Leu Ala 85 90 95 gag ttt ggc cag att gac gcc ttc att gcc aac gct ggc aag aca gcc 336 Glu Phe Gly Gln Ile Asp Ala Phe Ile Ala Asn Ala Gly Lys Thr Ala 100 105 110 agc agt ggt atc ctg gat ggt tcc gtt gag gac tgg gaa gag gtc atc 384 Ser Ser Gly Ile Leu Asp Gly Ser Val Glu Asp Trp Glu Glu Val Ile 115 120 125 cag aca gac ctg acg ggt acc ttc cac tgc gcc aag gcg gtg ggc ccg 432 Gln Thr Asp Leu Thr Gly Thr Phe His Cys Ala Lys Ala Val Gly Pro 130 135 140 cac ttc aag cag cgc gga acg ggc agc ttc atc atc acc tcc agc atg 480 His Phe Lys Gln Arg Gly Thr Gly Ser Phe Ile Ile Thr Ser Ser Met 145 150 155 160 tcg ggt cac att gcc aac ttc ccg cag gag cag acg tcg tac aac gtt 528 Ser Gly His Ile Ala Asn Phe Pro Gln Glu Gln Thr Ser Tyr Asn Val 165 170 175 gcc aag gca ggc tgc atc cat atg gcc cgg tca ttg gcc aac gag tgg 576 Ala Lys Ala Gly Cys Ile His Met Ala Arg Ser Leu Ala Asn Glu Trp 180 185 190 agg gac ttt gcc cgc gtg aac agc atc tcc ccg ggt tac atc gac acg 624 Arg Asp Phe Ala Arg Val Asn Ser Ile Ser Pro Gly Tyr Ile Asp Thr 195 200 205 ggg ctg tcg gac ttt gtg gat aag aag act cag gat ctg tgg atg tcg 672 Gly Leu Ser Asp Phe Val Asp Lys Lys Thr Gln Asp Leu Trp Met Ser 210 215 220 atg atc ccc atg gga cgc aac ggt gat gcc aag gag ctg aag ggt gca 720 Met Ile Pro Met Gly Arg Asn Gly Asp Ala Lys Glu Leu Lys Gly Ala 225 230 235 240 tat gtc tat ctc gcc agt gat gcc agc aca tac acg acg ggt gcc gat 768 Tyr Val Tyr Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp 245 250 255 ttg gtc att gac gga gga tac acc gtg cgg taa 801 Leu Val Ile Asp Gly Gly Tyr Thr Val Arg 260 265 <210> SEQ ID NO 26 <211> LENGTH: 1602 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1602) <400> SEQUENCE: 26 atg agt ggg gta agc ttt gcc cgt cag gcc tta ctc cag gct gct ctt 48 Met Ser Gly Val Ser Phe Ala Arg Gln Ala Leu Leu Gln Ala Ala Leu 1 5 10 15 ctt tcg att gtc tcc gca atc ccg gcg ccc acg gcc ttc gtc aac aat 96 Leu Ser Ile Val Ser Ala Ile Pro Ala Pro Thr Ala Phe Val Asn Asn 20 25 30 gtc gct ccg ccc gag ccc atc atc acc cct tcg ccg gtt caa cat cga 144 Val Ala Pro Pro Glu Pro Ile Ile Thr Pro Ser Pro Val Gln His Arg 35 40 45 cct tct cga gtc gct ggt cga aac att ctg agt gac gtc gat tct gat 192 Pro Ser Arg Val Ala Gly Arg Asn Ile Leu Ser Asp Val Asp Ser Asp 50 55 60 att aac agc atc ctt tct ggc ctt gga tcc gac cta ccc tcc tgg gtc 240 Ile Asn Ser Ile Leu Ser Gly Leu Gly Ser Asp Leu Pro Ser Trp Val 65 70 75 80 gca tcg ggt gtg ccc aac tac ttc cag ggt ttc cct act ggg gat gca 288 Ala Ser Gly Val Pro Asn Tyr Phe Gln Gly Phe Pro Thr Gly Asp Ala 85 90 95 gtg gtg agc tcc ttg ggc ttg aac agc gct gag ttg gca gcc ttg ccc 336 Val Val Ser Ser Leu Gly Leu Asn Ser Ala Glu Leu Ala Ala Leu Pro 100 105 110 acc aat gtc ttg aat atc gac cca tat gcc aac tgg act agc tct ggc 384 Thr Asn Val Leu Asn Ile Asp Pro Tyr Ala Asn Trp Thr Ser Ser Gly 115 120 125 tgg aac gtt cgc ttc cac gga aac gtc tac aag cag ccc aac acc tcc 432 Trp Asn Val Arg Phe His Gly Asn Val Tyr Lys Gln Pro Asn Thr Ser 130 135 140 atc tcc gac ctc aat gat ttg gct gat gtt ttc ctc ggc aat aca agc 480 Ile Ser Asp Leu Asn Asp Leu Ala Asp Val Phe Leu Gly Asn Thr Ser 145 150 155 160 atc tcc gac ctt tcc gaa tcc gag cag aaa cag gct cgg aat tta aca 528 Ile Ser Asp Leu Ser Glu Ser Glu Gln Lys Gln Ala Arg Asn Leu Thr 165 170 175 gcg gaa ata tta gtg gtc cag caa gct cac gtc gcc gta aac aca atc 576 Ala Glu Ile Leu Val Val Gln Gln Ala His Val Ala Val Asn Thr Ile 180 185 190 cac ctg gag cct gca ccg agt cag gga tcc agc gga cag tca ggc ggc 624 His Leu Glu Pro Ala Pro Ser Gln Gly Ser Ser Gly Gln Ser Gly Gly 195 200 205 ggt gga tcc tct aat acc act ggc ggc acc caa gac ctc act ctg ccc 672 Gly Gly Ser Ser Asn Thr Thr Gly Gly Thr Gln Asp Leu Thr Leu Pro 210 215 220 tac aac act aca gtt gaa ggc gat ttt gac act ttc gta ccg att agc 720 Tyr Asn Thr Thr Val Glu Gly Asp Phe Asp Thr Phe Val Pro Ile Ser 225 230 235 240 agc aat ggc ttg acg gca ggt aat gaa act tcg gcg ata caa cgg ctt 768 Ser Asn Gly Leu Thr Ala Gly Asn Glu Thr Ser Ala Ile Gln Arg Leu 245 250 255 aac gtc cat gtg gag ggc gcg aca ata gga aac agt act gcc tat ctc 816 Asn Val His Val Glu Gly Ala Thr Ile Gly Asn Ser Thr Ala Tyr Leu 260 265 270 gta cct ccg acc ggg ctc act gtc gtt tcg gac atc gat gat atc cta 864 Val Pro Pro Thr Gly Leu Thr Val Val Ser Asp Ile Asp Asp Ile Leu 275 280 285 cgc gtc acc aaa atc tat gaa cca gag caa ggt tta ctc aac tca ttt 912 Arg Val Thr Lys Ile Tyr Glu Pro Glu Gln Gly Leu Leu Asn Ser Phe 290 295 300 gcg cgc ccc ttc agg ccg tgg gag aat atg ccg gac atc tat cgc aac 960 Ala Arg Pro Phe Arg Pro Trp Glu Asn Met Pro Asp Ile Tyr Arg Asn 305 310 315 320 tgg tcg atc agt ctc ccc aac ctg cac ttt cac tac cta acg acg acc 1008 Trp Ser Ile Ser Leu Pro Asn Leu His Phe His Tyr Leu Thr Thr Thr 325 330 335 ccc gaa caa gtc acc agg aac tac atg caa ttc atc tac gac aac tac 1056 Pro Glu Gln Val Thr Arg Asn Tyr Met Gln Phe Ile Tyr Asp Asn Tyr 340 345 350 ccc ggt gga tct ttc gac acc cgt ccc ctc aac ttt agc gac gtt tcc 1104 Pro Gly Gly Ser Phe Asp Thr Arg Pro Leu Asn Phe Ser Asp Val Ser 355 360 365 gcc acc ttg tcg atc cgg aaa ttc ctc ctg caa aag gtc ttc gag acc 1152 Ala Thr Leu Ser Ile Arg Lys Phe Leu Leu Gln Lys Val Phe Glu Thr 370 375 380 ttt ccg cag cgc aag ttc atc ctc atc gcc gac acc agc aac agc gac 1200 Phe Pro Gln Arg Lys Phe Ile Leu Ile Ala Asp Thr Ser Asn Ser Asp 385 390 395 400 gtc atg cgt gac tat cct gaa atg gcg acc gac ttc cct ggt caa gtg 1248 Val Met Arg Asp Tyr Pro Glu Met Ala Thr Asp Phe Pro Gly Gln Val 405 410 415 caa tgt att ttt ctc cgg aac acc agc gcc acc gac tca ggg gac aaa 1296 Gln Cys Ile Phe Leu Arg Asn Thr Ser Ala Thr Asp Ser Gly Asp Lys 420 425 430 ttc cca tac gat acc tct ggg ttt aag aag ctc aat cag tca aac tac 1344 Phe Pro Tyr Asp Thr Ser Gly Phe Lys Lys Leu Asn Gln Ser Asn Tyr 435 440 445 atg ttc ttc ttg cat ccc gat gac ttg acg aat cta gac atc gcg aat 1392 Met Phe Phe Leu His Pro Asp Asp Leu Thr Asn Leu Asp Ile Ala Asn 450 455 460 ggc cag tgt tac aac aca tcc att ccg cag aat tta acg ttt agt tat 1440 Gly Gln Cys Tyr Asn Thr Ser Ile Pro Gln Asn Leu Thr Phe Ser Tyr 465 470 475 480 cag ggt ttg cca ttg ggg ctg ggt gat gaa ccc aca gct gtc aat ggg 1488 Gln Gly Leu Pro Leu Gly Leu Gly Asp Glu Pro Thr Ala Val Asn Gly 485 490 495 tct gcc aat cat act gct gag tcg gcg gcc ggt ttg tcg gta aag ggt 1536 Ser Ala Asn His Thr Ala Glu Ser Ala Ala Gly Leu Ser Val Lys Gly 500 505 510 ggg act gat atg cag ggt ttg atc ttt ctg ttc acc ctg gtt acg gcc 1584 Gly Thr Asp Met Gln Gly Leu Ile Phe Leu Phe Thr Leu Val Thr Ala 515 520 525 tat tcc att ttc ttg taa 1602 Tyr Ser Ile Phe Leu 530 <210> SEQ ID NO 27 <211> LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1392) <400> SEQUENCE: 27 atg tcg aag tcg aat ggc gtc agc ctg gca ttc ccc gcc gaa gca gcg 48 Met Ser Lys Ser Asn Gly Val Ser Leu Ala Phe Pro Ala Glu Ala Ala 1 5 10 15 aca aag gaa tat gca gca tct cta gac tct tct gat aga ctt gct gca 96 Thr Lys Glu Tyr Ala Ala Ser Leu Asp Ser Ser Asp Arg Leu Ala Ala 20 25 30 ttt cgg gag aag ttc atc gtc cca tcg aaa gca aat att gct tcc aaa 144 Phe Arg Glu Lys Phe Ile Val Pro Ser Lys Ala Asn Ile Ala Ser Lys 35 40 45 aag cta gca aag cct ggc ctt tcg ccc gag tca tgc att tac ttc tgc 192 Lys Leu Ala Lys Pro Gly Leu Ser Pro Glu Ser Cys Ile Tyr Phe Cys 50 55 60 ggt aac tca ctt ggc att caa ccc aag gcc aca gca aag tac tta gaa 240 Gly Asn Ser Leu Gly Ile Gln Pro Lys Ala Thr Ala Lys Tyr Leu Glu 65 70 75 80 gca cag ttg gac act tgg tca tct att gga gtt ggc ggc cac ttc act 288 Ala Gln Leu Asp Thr Trp Ser Ser Ile Gly Val Gly Gly His Phe Thr 85 90 95 gat ctt gag ggc tca ccc ttg aag caa tgg cag ctg ctc tca gag caa 336 Asp Leu Glu Gly Ser Pro Leu Lys Gln Trp Gln Leu Leu Ser Glu Gln 100 105 110 gca gct gat tca atg agc aag att gtg gga gca aag cca gaa gaa gtc 384 Ala Ala Asp Ser Met Ser Lys Ile Val Gly Ala Lys Pro Glu Glu Val 115 120 125 gca gcg atg gga aca ctt aca acg aac ctt cat ctt ctg ctg gct agt 432 Ala Ala Met Gly Thr Leu Thr Thr Asn Leu His Leu Leu Leu Ala Ser 130 135 140 ttc tat aaa ccg act caa aca aag cac aag atc ttg atg gat tgg aag 480 Phe Tyr Lys Pro Thr Gln Thr Lys His Lys Ile Leu Met Asp Trp Lys 145 150 155 160 gct ttc cca agt gat cac tac gct atc gaa tct cat att gcc tgg cat 528 Ala Phe Pro Ser Asp His Tyr Ala Ile Glu Ser His Ile Ala Trp His 165 170 175 gac ctg gat cct aag gag tct atg gtg ctc atc ggt cca gat gaa ggc 576 Asp Leu Asp Pro Lys Glu Ser Met Val Leu Ile Gly Pro Asp Glu Gly 180 185 190 gaa tac gag ata tcc acc cag aag att ttc tct tat atc gac aag cat 624 Glu Tyr Glu Ile Ser Thr Gln Lys Ile Phe Ser Tyr Ile Asp Lys His 195 200 205 gct gat gag gct gct atg att ctt ctt cct ggt att caa tat tat act 672 Ala Asp Glu Ala Ala Met Ile Leu Leu Pro Gly Ile Gln Tyr Tyr Thr 210 215 220 ggg caa ctg ttc gat atc cag aaa att aca aaa tat gcc cat tcg cgc 720 Gly Gln Leu Phe Asp Ile Gln Lys Ile Thr Lys Tyr Ala His Ser Arg 225 230 235 240 aac atg gtt gtt ggc tgg gat ctg gcc cat gcg ttc gct aat gtt gag 768 Asn Met Val Val Gly Trp Asp Leu Ala His Ala Phe Ala Asn Val Glu 245 250 255 cta aag ctg cat gat tgg aac gtt gat ttt gca gca tgg tgc aca tat 816 Leu Lys Leu His Asp Trp Asn Val Asp Phe Ala Ala Trp Cys Thr Tyr 260 265 270 aag tac gga aac gcc ggt cct gga gca atg ggg ggg ctt ttc gtg cat 864 Lys Tyr Gly Asn Ala Gly Pro Gly Ala Met Gly Gly Leu Phe Val His 275 280 285 gaa caa cac gga gag gtc gat tac agc gcg gga gaa gat gca ccc aaa 912 Glu Gln His Gly Glu Val Asp Tyr Ser Ala Gly Glu Asp Ala Pro Lys 290 295 300 ttc cgc cat cgt ctg acc ggg tgg tat ggt ggc gat cga tcg gta aga 960 Phe Arg His Arg Leu Thr Gly Trp Tyr Gly Gly Asp Arg Ser Val Arg 305 310 315 320 ttc aag atg gac aac aag ttc aaa cct att cct ggg gca gga gga ttt 1008 Phe Lys Met Asp Asn Lys Phe Lys Pro Ile Pro Gly Ala Gly Gly Phe 325 330 335 cag ata tca aat cct tcg gcc atc gac ctt gcg tgt ctt tgt gcc gct 1056 Gln Ile Ser Asn Pro Ser Ala Ile Asp Leu Ala Cys Leu Cys Ala Ala 340 345 350 tta tcg gtg ttt gat gag acg tca atg gca gat ctt cgc aga aaa tca 1104 Leu Ser Val Phe Asp Glu Thr Ser Met Ala Asp Leu Arg Arg Lys Ser 355 360 365 ttg aag cta aca gca tac ctt gag ttc ctt ttg ctc agg gat tat gaa 1152 Leu Lys Leu Thr Ala Tyr Leu Glu Phe Leu Leu Leu Arg Asp Tyr Glu 370 375 380 gaa gaa tct agg cca ttc agc att atc acg ccc aag gac ccc gag gcg 1200 Glu Glu Ser Arg Pro Phe Ser Ile Ile Thr Pro Lys Asp Pro Glu Ala 385 390 395 400 aga ggc gca caa ctg agt cta ttg ctc aag cct ggt cta tta cag aat 1248 Arg Gly Ala Gln Leu Ser Leu Leu Leu Lys Pro Gly Leu Leu Gln Asn 405 410 415 gtt gca cag aag ttg caa gaa gca gga ata gtc tgc gat aaa cgg gag 1296 Val Ala Gln Lys Leu Gln Glu Ala Gly Ile Val Cys Asp Lys Arg Glu 420 425 430 ccg ggt gtt gtg cgt gtc gct ccg gtt cct ttg tac aac agc ttc agc 1344 Pro Gly Val Val Arg Val Ala Pro Val Pro Leu Tyr Asn Ser Phe Ser 435 440 445 gag gtt tgg aca ttt gtg aag atc ttc aag gat gca ctg cag cag tga 1392 Glu Val Trp Thr Phe Val Lys Ile Phe Lys Asp Ala Leu Gln Gln 450 455 460 <210> SEQ ID NO 28 <211> LENGTH: 1143 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1143) <400> SEQUENCE: 28 atg gat gtg act aat ttg gct gtc gat tgt ccc gtt ata gag acc ggt 48 Met Asp Val Thr Asn Leu Ala Val Asp Cys Pro Val Ile Glu Thr Gly 1 5 10 15 tcc ctt gct ttg tgg atg gct tcc tcc gaa gtg gac cag aag gcc ttg 96 Ser Leu Ala Leu Trp Met Ala Ser Ser Glu Val Asp Gln Lys Ala Leu 20 25 30 ggc aac ttt gcg gcc ata acg ggc ctc gag aac ccg ttc ctg tcc gcg 144 Gly Asn Phe Ala Ala Ile Thr Gly Leu Glu Asn Pro Phe Leu Ser Ala 35 40 45 atg ctg tac aag att ctg ggg ttg tct gtt atg tta tcg aag aaa ctc 192 Met Leu Tyr Lys Ile Leu Gly Leu Ser Val Met Leu Ser Lys Lys Leu 50 55 60 ctc cgt gca cga cga ttg cgg cgc cta gac ccc acc cga gag act aaa 240 Leu Arg Ala Arg Arg Leu Arg Arg Leu Asp Pro Thr Arg Glu Thr Lys 65 70 75 80 tcg ctt cat ctt tat tac cat atc atc tgg ctt tcg cgt gag ggt ctt 288 Ser Leu His Leu Tyr Tyr His Ile Ile Trp Leu Ser Arg Glu Gly Leu 85 90 95 ctg ata tta gag gag ttc gtc ctg cca ctg gtt gag gga ttt atg gaa 336 Leu Ile Leu Glu Glu Phe Val Leu Pro Leu Val Glu Gly Phe Met Glu 100 105 110 ctc aaa att tta gct tac aag ctg cgc gcc tcc ttt tac cat ata ttc 384 Leu Lys Ile Leu Ala Tyr Lys Leu Arg Ala Ser Phe Tyr His Ile Phe 115 120 125 gtc cta ttt cag aac cag cct gct gtc cac tcg ccg ggt atc gtt agt 432 Val Leu Phe Gln Asn Gln Pro Ala Val His Ser Pro Gly Ile Val Ser 130 135 140 ctg ccg agt agt aca cct tta tcc aac ggc gtg aca gaa gcg gag tca 480 Leu Pro Ser Ser Thr Pro Leu Ser Asn Gly Val Thr Glu Ala Glu Ser 145 150 155 160 cca tcc aag gat tcc aat ttg aga ttt tca ttt cag ctg aag ccc gac 528 Pro Ser Lys Asp Ser Asn Leu Arg Phe Ser Phe Gln Leu Lys Pro Asp 165 170 175 acg ata acg gtt tcc ggg aag ccc agt tcc gcc tcg gat agt gcg cca 576 Thr Ile Thr Val Ser Gly Lys Pro Ser Ser Ala Ser Asp Ser Ala Pro 180 185 190 cga ggc aag gtt acg cag gca ccc ccg ggc ttt gcg ccg gtt caa ccg 624 Arg Gly Lys Val Thr Gln Ala Pro Pro Gly Phe Ala Pro Val Gln Pro 195 200 205 ccc aag tcg aac tcg gcg ttt ctc ctc cct gct ctc gat tat acc cct 672 Pro Lys Ser Asn Ser Ala Phe Leu Leu Pro Ala Leu Asp Tyr Thr Pro 210 215 220 aca gct acc gca tgc ttc aat cat gcc gcc ctc ttg gca gat caa ttc 720 Thr Ala Thr Ala Cys Phe Asn His Ala Ala Leu Leu Ala Asp Gln Phe 225 230 235 240 ctc ccg ggg tcg cac ccg ctc cgt ctg tcc atc aag ctt gaa ttt gcc 768 Leu Pro Gly Ser His Pro Leu Arg Leu Ser Ile Lys Leu Glu Phe Ala 245 250 255 gcc tac ctc tac gat tgc cta cat gac gct cat gcc tgt cga cgg ttg 816 Ala Tyr Leu Tyr Asp Cys Leu His Asp Ala His Ala Cys Arg Arg Leu 260 265 270 gcc aag caa gcc att gcc gat gtg tac aat gcg caa gag ggc atg gac 864 Ala Lys Gln Ala Ile Ala Asp Val Tyr Asn Ala Gln Glu Gly Met Asp 275 280 285 gac gag agc ttc gag gat gca gct gag att gtg ggt att ttg ggc aag 912 Asp Glu Ser Phe Glu Asp Ala Ala Glu Ile Val Gly Ile Leu Gly Lys 290 295 300 atg gtg aag cgg gga agg aac aca agc agt gct gga aat agc acc acg 960 Met Val Lys Arg Gly Arg Asn Thr Ser Ser Ala Gly Asn Ser Thr Thr 305 310 315 320 gct gtg aaa acg ccc cag gag gag cga agt gag ggc agt cga aca cca 1008 Ala Val Lys Thr Pro Gln Glu Glu Arg Ser Glu Gly Ser Arg Thr Pro 325 330 335 tcc tct cag aca acg ttg aaa agg ccg gcg cga gtg ccg aaa agc acc 1056 Ser Ser Gln Thr Thr Leu Lys Arg Pro Ala Arg Val Pro Lys Ser Thr 340 345 350 tcc tca ccg gcg cgc gcg acc aag ccg acg act agc gag ggg atg tcc 1104 Ser Ser Pro Ala Arg Ala Thr Lys Pro Thr Thr Ser Glu Gly Met Ser 355 360 365 ccc gct gtg ccc gac ccc acc atg atg aat ccc ata tga 1143 Pro Ala Val Pro Asp Pro Thr Met Met Asn Pro Ile 370 375 380 <210> SEQ ID NO 29 <211> LENGTH: 1779 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1779) <400> SEQUENCE: 29 atg aga aat ttg gag tgt gtc tac aca aaa tca cga agg tat ccc tcg 48 Met Arg Asn Leu Glu Cys Val Tyr Thr Lys Ser Arg Arg Tyr Pro Ser 1 5 10 15 tct atc ttt ccg aca ctc agg agg tcg tca act agt ccg aac acg gcg 96 Ser Ile Phe Pro Thr Leu Arg Arg Ser Ser Thr Ser Pro Asn Thr Ala 20 25 30 tcc tct cct gac act aat gcc tca tct tcg cca tgc tcg gac tct agt 144 Ser Ser Pro Asp Thr Asn Ala Ser Ser Ser Pro Cys Ser Asp Ser Ser 35 40 45 ggg acc cat gct ggc att tcc att ccc cac tca gat agt ccc gcc agt 192 Gly Thr His Ala Gly Ile Ser Ile Pro His Ser Asp Ser Pro Ala Ser 50 55 60 gtg gac gaa tgt ggg aac aac ggc ctt ttc gac ggg gaa tct cta act 240 Val Asp Glu Cys Gly Asn Asn Gly Leu Phe Asp Gly Glu Ser Leu Thr 65 70 75 80 caa tta ttg gtc tct ggg agc aaa gcc ata tca ggc caa cca cat atc 288 Gln Leu Leu Val Ser Gly Ser Lys Ala Ile Ser Gly Gln Pro His Ile 85 90 95 gcc att aag cca cat gtt ctg tct acc tat ccc ctt gac aac ctc ctc 336 Ala Ile Lys Pro His Val Leu Ser Thr Tyr Pro Leu Asp Asn Leu Leu 100 105 110 tca gac att gag gga ctc ctc gag cag cca gtc tct cat ttg gct caa 384 Ser Asp Ile Glu Gly Leu Leu Glu Gln Pro Val Ser His Leu Ala Gln 115 120 125 agc aag gaa gat cta aca aaa ccg gac cgg atg ttt cga act cgc ctt 432 Ser Lys Glu Asp Leu Thr Lys Pro Asp Arg Met Phe Arg Thr Arg Leu 130 135 140 cca gcg gtc tca ggt gag gac aat gag tat ctg cta agg aaa gga gct 480 Pro Ala Val Ser Gly Glu Asp Asn Glu Tyr Leu Leu Arg Lys Gly Ala 145 150 155 160 ctg ctg ctc ttg cca cca gcc ttg cgc aac gaa cta ttg gcg gca tat 528 Leu Leu Leu Leu Pro Pro Ala Leu Arg Asn Glu Leu Leu Ala Ala Tyr 165 170 175 gtc aac tac gtc cat ccg cta tta ccg att ctt gat ctt gga gac ttt 576 Val Asn Tyr Val His Pro Leu Leu Pro Ile Leu Asp Leu Gly Asp Phe 180 185 190 ctc tca caa atc att gtt gga gat gat gct caa ttc aag tca cca cta 624 Leu Ser Gln Ile Ile Val Gly Asp Asp Ala Gln Phe Lys Ser Pro Leu 195 200 205 tta tac caa gct gtc atg ttc gcg ggc agt att ttt gct cag cga gac 672 Leu Tyr Gln Ala Val Met Phe Ala Gly Ser Ile Phe Ala Gln Arg Asp 210 215 220 ggc aca gac gaa cct gaa cag cta acg agg gct tta ttt gaa cgt aca 720 Gly Thr Asp Glu Pro Glu Gln Leu Thr Arg Ala Leu Phe Glu Arg Thr 225 230 235 240 aag gtt cta tac gag ttt gaa tcc gaa tca tgt gcc tac act caa att 768 Lys Val Leu Tyr Glu Phe Glu Ser Glu Ser Cys Ala Tyr Thr Gln Ile 245 250 255 caa gct ctc tta ttg atg act ctc tgg cat gga gat atg tca cgc cac 816 Gln Ala Leu Leu Leu Met Thr Leu Trp His Gly Asp Met Ser Arg His 260 265 270 aaa ggc ccg tct tac tgg ctt gac gtc gcc ttc tcc act gcg gaa aga 864 Lys Gly Pro Ser Tyr Trp Leu Asp Val Ala Phe Ser Thr Ala Glu Arg 275 280 285 att ggc cta ctc cat gat gaa gac tgg cca tgc gat agc cgt tcc ttc 912 Ile Gly Leu Leu His Asp Glu Asp Trp Pro Cys Asp Ser Arg Ser Phe 290 295 300 cga tcc gct atg tgg tgt tgc ctt tac gta cgt gat cga aca atc tcg 960 Arg Ser Ala Met Trp Cys Cys Leu Tyr Val Arg Asp Arg Thr Ile Ser 305 310 315 320 ctt ggg tct cgt cgg cct cca cgg atg cca gtc aac aaa tat ccg gac 1008 Leu Gly Ser Arg Arg Pro Pro Arg Met Pro Val Asn Lys Tyr Pro Asp 325 330 335 tca att ctc cat ctt atg agc gac gca cct cgg gaa tat gac gaa ata 1056 Ser Ile Leu His Leu Met Ser Asp Ala Pro Arg Glu Tyr Asp Glu Ile 340 345 350 att ctt gag agt cta ggt agc gct tct ctg atg ctt aca agc act tac 1104 Ile Leu Glu Ser Leu Gly Ser Ala Ser Leu Met Leu Thr Ser Thr Tyr 355 360 365 caa gaa caa tcg acc atg ctc ttc aag gaa ctt gtc aaa ctc tcc tac 1152 Gln Glu Gln Ser Thr Met Leu Phe Lys Glu Leu Val Lys Leu Ser Tyr 370 375 380 tgc gtc ggg gca atc tta gat tat cta tac gaa ggg gcc tgg gtc cga 1200 Cys Val Gly Ala Ile Leu Asp Tyr Leu Tyr Glu Gly Ala Trp Val Arg 385 390 395 400 ata cca aca cgt cgc agc agc ttt tat tca ctc gcc cct aaa tgc agc 1248 Ile Pro Thr Arg Arg Ser Ser Phe Tyr Ser Leu Ala Pro Lys Cys Ser 405 410 415 atc tcg cca tcc atc aga cca agc tgt gag aaa cta ctt tac tct tgg 1296 Ile Ser Pro Ser Ile Arg Pro Ser Cys Glu Lys Leu Leu Tyr Ser Trp 420 425 430 att caa ttc ctc cct ctc aac gca gtc tac cat ccg cca cag ttg ccc 1344 Ile Gln Phe Leu Pro Leu Asn Ala Val Tyr His Pro Pro Gln Leu Pro 435 440 445 gcc gat gcg gaa ggg gag tca gcc gaa aca gtc ttc ctt gtc cac aaa 1392 Ala Asp Ala Glu Gly Glu Ser Ala Glu Thr Val Phe Leu Val His Lys 450 455 460 gcc ttc ctt tac ttg ctg tac cta gcc tcc atg gca gtc ata tac cgc 1440 Ala Phe Leu Tyr Leu Leu Tyr Leu Ala Ser Met Ala Val Ile Tyr Arg 465 470 475 480 aca ggc act cat agc caa caa acc agc acg gca act ccg aac atg gaa 1488 Thr Gly Thr His Ser Gln Gln Thr Ser Thr Ala Thr Pro Asn Met Glu 485 490 495 ggt cta cgg gca tcg acg tca caa ata aaa aag gtc ctt gcg gag ctg 1536 Gly Leu Arg Ala Ser Thr Ser Gln Ile Lys Lys Val Leu Ala Glu Leu 500 505 510 cag ggc atg gga ctg ctt cag ttc ctt cca ggg gca tca gtc act atc 1584 Gln Gly Met Gly Leu Leu Gln Phe Leu Pro Gly Ala Ser Val Thr Ile 515 520 525 ctg atg ttc gct gtg gag gct agt ctg ttg gat ctc cag ggc tcg gat 1632 Leu Met Phe Ala Val Glu Ala Ser Leu Leu Asp Leu Gln Gly Ser Asp 530 535 540 acc atg gtc cga agg cag gct atg tca aat cta tat gcc tgt gag gag 1680 Thr Met Val Arg Arg Gln Ala Met Ser Asn Leu Tyr Ala Cys Glu Glu 545 550 555 560 gct gcg tta cac ttg atg cag gct tat cct acg gct gag atg gcg gta 1728 Ala Ala Leu His Leu Met Gln Ala Tyr Pro Thr Ala Glu Met Ala Val 565 570 575 ttg aag act agg aca gcc cgt gca aac ctt ctt ggg ttg gtt gag aca 1776 Leu Lys Thr Arg Thr Ala Arg Ala Asn Leu Leu Gly Leu Val Glu Thr 580 585 590 tga 1779 <210> SEQ ID NO 30 <211> LENGTH: 266 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 30 Met Pro Ile Ser Ile Pro Ser Ala Ser Ser Val His Asp Leu Phe Ser 1 5 10 15 Leu Lys Gly Lys Val Val Val Ile Thr Gly Ala Ser Gly Pro Arg Gly 20 25 30 Met Gly Ile Glu Ala Ala Arg Gly Cys Ala Glu Met Gly Ala Asn Ile 35 40 45 Ala Leu Thr Tyr Ser Ser Arg Pro Gln Gly Gly Glu Lys Asn Ala Glu 50 55 60 Glu Leu Arg Asn Thr Tyr Gly Val Lys Ala Lys Ala Tyr Gln Cys Asn 65 70 75 80 Val Gly Asp Trp Asn Ser Val Lys Lys Leu Val Asp Asp Val Leu Ala 85 90 95 Glu Phe Gly Gln Ile Asp Ala Phe Ile Ala Asn Ala Gly Lys Thr Ala 100 105 110 Ser Ser Gly Ile Leu Asp Gly Ser Val Glu Asp Trp Glu Glu Val Ile 115 120 125 Gln Thr Asp Leu Thr Gly Thr Phe His Cys Ala Lys Ala Val Gly Pro 130 135 140 His Phe Lys Gln Arg Gly Thr Gly Ser Phe Ile Ile Thr Ser Ser Met 145 150 155 160 Ser Gly His Ile Ala Asn Phe Pro Gln Glu Gln Thr Ser Tyr Asn Val 165 170 175 Ala Lys Ala Gly Cys Ile His Met Ala Arg Ser Leu Ala Asn Glu Trp 180 185 190 Arg Asp Phe Ala Arg Val Asn Ser Ile Ser Pro Gly Tyr Ile Asp Thr 195 200 205 Gly Leu Ser Asp Phe Val Asp Lys Lys Thr Gln Asp Leu Trp Met Ser 210 215 220 Met Ile Pro Met Gly Arg Asn Gly Asp Ala Lys Glu Leu Lys Gly Ala 225 230 235 240 Tyr Val Tyr Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp 245 250 255 Leu Val Ile Asp Gly Gly Tyr Thr Val Arg 260 265 <210> SEQ ID NO 31 <211> LENGTH: 533 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 31 Met Ser Gly Val Ser Phe Ala Arg Gln Ala Leu Leu Gln Ala Ala Leu 1 5 10 15 Leu Ser Ile Val Ser Ala Ile Pro Ala Pro Thr Ala Phe Val Asn Asn 20 25 30 Val Ala Pro Pro Glu Pro Ile Ile Thr Pro Ser Pro Val Gln His Arg 35 40 45 Pro Ser Arg Val Ala Gly Arg Asn Ile Leu Ser Asp Val Asp Ser Asp 50 55 60 Ile Asn Ser Ile Leu Ser Gly Leu Gly Ser Asp Leu Pro Ser Trp Val 65 70 75 80 Ala Ser Gly Val Pro Asn Tyr Phe Gln Gly Phe Pro Thr Gly Asp Ala 85 90 95 Val Val Ser Ser Leu Gly Leu Asn Ser Ala Glu Leu Ala Ala Leu Pro 100 105 110 Thr Asn Val Leu Asn Ile Asp Pro Tyr Ala Asn Trp Thr Ser Ser Gly 115 120 125 Trp Asn Val Arg Phe His Gly Asn Val Tyr Lys Gln Pro Asn Thr Ser 130 135 140 Ile Ser Asp Leu Asn Asp Leu Ala Asp Val Phe Leu Gly Asn Thr Ser 145 150 155 160 Ile Ser Asp Leu Ser Glu Ser Glu Gln Lys Gln Ala Arg Asn Leu Thr 165 170 175 Ala Glu Ile Leu Val Val Gln Gln Ala His Val Ala Val Asn Thr Ile 180 185 190 His Leu Glu Pro Ala Pro Ser Gln Gly Ser Ser Gly Gln Ser Gly Gly 195 200 205 Gly Gly Ser Ser Asn Thr Thr Gly Gly Thr Gln Asp Leu Thr Leu Pro 210 215 220 Tyr Asn Thr Thr Val Glu Gly Asp Phe Asp Thr Phe Val Pro Ile Ser 225 230 235 240 Ser Asn Gly Leu Thr Ala Gly Asn Glu Thr Ser Ala Ile Gln Arg Leu 245 250 255 Asn Val His Val Glu Gly Ala Thr Ile Gly Asn Ser Thr Ala Tyr Leu 260 265 270 Val Pro Pro Thr Gly Leu Thr Val Val Ser Asp Ile Asp Asp Ile Leu 275 280 285 Arg Val Thr Lys Ile Tyr Glu Pro Glu Gln Gly Leu Leu Asn Ser Phe 290 295 300 Ala Arg Pro Phe Arg Pro Trp Glu Asn Met Pro Asp Ile Tyr Arg Asn 305 310 315 320 Trp Ser Ile Ser Leu Pro Asn Leu His Phe His Tyr Leu Thr Thr Thr 325 330 335 Pro Glu Gln Val Thr Arg Asn Tyr Met Gln Phe Ile Tyr Asp Asn Tyr 340 345 350 Pro Gly Gly Ser Phe Asp Thr Arg Pro Leu Asn Phe Ser Asp Val Ser 355 360 365 Ala Thr Leu Ser Ile Arg Lys Phe Leu Leu Gln Lys Val Phe Glu Thr 370 375 380 Phe Pro Gln Arg Lys Phe Ile Leu Ile Ala Asp Thr Ser Asn Ser Asp 385 390 395 400 Val Met Arg Asp Tyr Pro Glu Met Ala Thr Asp Phe Pro Gly Gln Val 405 410 415 Gln Cys Ile Phe Leu Arg Asn Thr Ser Ala Thr Asp Ser Gly Asp Lys 420 425 430 Phe Pro Tyr Asp Thr Ser Gly Phe Lys Lys Leu Asn Gln Ser Asn Tyr 435 440 445 Met Phe Phe Leu His Pro Asp Asp Leu Thr Asn Leu Asp Ile Ala Asn 450 455 460 Gly Gln Cys Tyr Asn Thr Ser Ile Pro Gln Asn Leu Thr Phe Ser Tyr 465 470 475 480 Gln Gly Leu Pro Leu Gly Leu Gly Asp Glu Pro Thr Ala Val Asn Gly 485 490 495 Ser Ala Asn His Thr Ala Glu Ser Ala Ala Gly Leu Ser Val Lys Gly 500 505 510 Gly Thr Asp Met Gln Gly Leu Ile Phe Leu Phe Thr Leu Val Thr Ala 515 520 525 Tyr Ser Ile Phe Leu 530 <210> SEQ ID NO 32 <211> LENGTH: 463 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 32 Met Ser Lys Ser Asn Gly Val Ser Leu Ala Phe Pro Ala Glu Ala Ala 1 5 10 15 Thr Lys Glu Tyr Ala Ala Ser Leu Asp Ser Ser Asp Arg Leu Ala Ala 20 25 30 Phe Arg Glu Lys Phe Ile Val Pro Ser Lys Ala Asn Ile Ala Ser Lys 35 40 45 Lys Leu Ala Lys Pro Gly Leu Ser Pro Glu Ser Cys Ile Tyr Phe Cys 50 55 60 Gly Asn Ser Leu Gly Ile Gln Pro Lys Ala Thr Ala Lys Tyr Leu Glu 65 70 75 80 Ala Gln Leu Asp Thr Trp Ser Ser Ile Gly Val Gly Gly His Phe Thr 85 90 95 Asp Leu Glu Gly Ser Pro Leu Lys Gln Trp Gln Leu Leu Ser Glu Gln 100 105 110 Ala Ala Asp Ser Met Ser Lys Ile Val Gly Ala Lys Pro Glu Glu Val 115 120 125 Ala Ala Met Gly Thr Leu Thr Thr Asn Leu His Leu Leu Leu Ala Ser 130 135 140 Phe Tyr Lys Pro Thr Gln Thr Lys His Lys Ile Leu Met Asp Trp Lys 145 150 155 160 Ala Phe Pro Ser Asp His Tyr Ala Ile Glu Ser His Ile Ala Trp His 165 170 175 Asp Leu Asp Pro Lys Glu Ser Met Val Leu Ile Gly Pro Asp Glu Gly 180 185 190 Glu Tyr Glu Ile Ser Thr Gln Lys Ile Phe Ser Tyr Ile Asp Lys His 195 200 205 Ala Asp Glu Ala Ala Met Ile Leu Leu Pro Gly Ile Gln Tyr Tyr Thr 210 215 220 Gly Gln Leu Phe Asp Ile Gln Lys Ile Thr Lys Tyr Ala His Ser Arg 225 230 235 240 Asn Met Val Val Gly Trp Asp Leu Ala His Ala Phe Ala Asn Val Glu 245 250 255 Leu Lys Leu His Asp Trp Asn Val Asp Phe Ala Ala Trp Cys Thr Tyr 260 265 270 Lys Tyr Gly Asn Ala Gly Pro Gly Ala Met Gly Gly Leu Phe Val His 275 280 285 Glu Gln His Gly Glu Val Asp Tyr Ser Ala Gly Glu Asp Ala Pro Lys 290 295 300 Phe Arg His Arg Leu Thr Gly Trp Tyr Gly Gly Asp Arg Ser Val Arg 305 310 315 320 Phe Lys Met Asp Asn Lys Phe Lys Pro Ile Pro Gly Ala Gly Gly Phe 325 330 335 Gln Ile Ser Asn Pro Ser Ala Ile Asp Leu Ala Cys Leu Cys Ala Ala 340 345 350 Leu Ser Val Phe Asp Glu Thr Ser Met Ala Asp Leu Arg Arg Lys Ser 355 360 365 Leu Lys Leu Thr Ala Tyr Leu Glu Phe Leu Leu Leu Arg Asp Tyr Glu 370 375 380 Glu Glu Ser Arg Pro Phe Ser Ile Ile Thr Pro Lys Asp Pro Glu Ala 385 390 395 400 Arg Gly Ala Gln Leu Ser Leu Leu Leu Lys Pro Gly Leu Leu Gln Asn 405 410 415 Val Ala Gln Lys Leu Gln Glu Ala Gly Ile Val Cys Asp Lys Arg Glu 420 425 430 Pro Gly Val Val Arg Val Ala Pro Val Pro Leu Tyr Asn Ser Phe Ser 435 440 445 Glu Val Trp Thr Phe Val Lys Ile Phe Lys Asp Ala Leu Gln Gln 450 455 460 <210> SEQ ID NO 33 <211> LENGTH: 380 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 33 Met Asp Val Thr Asn Leu Ala Val Asp Cys Pro Val Ile Glu Thr Gly 1 5 10 15 Ser Leu Ala Leu Trp Met Ala Ser Ser Glu Val Asp Gln Lys Ala Leu 20 25 30 Gly Asn Phe Ala Ala Ile Thr Gly Leu Glu Asn Pro Phe Leu Ser Ala 35 40 45 Met Leu Tyr Lys Ile Leu Gly Leu Ser Val Met Leu Ser Lys Lys Leu 50 55 60 Leu Arg Ala Arg Arg Leu Arg Arg Leu Asp Pro Thr Arg Glu Thr Lys 65 70 75 80 Ser Leu His Leu Tyr Tyr His Ile Ile Trp Leu Ser Arg Glu Gly Leu 85 90 95 Leu Ile Leu Glu Glu Phe Val Leu Pro Leu Val Glu Gly Phe Met Glu 100 105 110 Leu Lys Ile Leu Ala Tyr Lys Leu Arg Ala Ser Phe Tyr His Ile Phe 115 120 125 Val Leu Phe Gln Asn Gln Pro Ala Val His Ser Pro Gly Ile Val Ser 130 135 140 Leu Pro Ser Ser Thr Pro Leu Ser Asn Gly Val Thr Glu Ala Glu Ser 145 150 155 160 Pro Ser Lys Asp Ser Asn Leu Arg Phe Ser Phe Gln Leu Lys Pro Asp 165 170 175 Thr Ile Thr Val Ser Gly Lys Pro Ser Ser Ala Ser Asp Ser Ala Pro 180 185 190 Arg Gly Lys Val Thr Gln Ala Pro Pro Gly Phe Ala Pro Val Gln Pro 195 200 205 Pro Lys Ser Asn Ser Ala Phe Leu Leu Pro Ala Leu Asp Tyr Thr Pro 210 215 220 Thr Ala Thr Ala Cys Phe Asn His Ala Ala Leu Leu Ala Asp Gln Phe 225 230 235 240 Leu Pro Gly Ser His Pro Leu Arg Leu Ser Ile Lys Leu Glu Phe Ala 245 250 255 Ala Tyr Leu Tyr Asp Cys Leu His Asp Ala His Ala Cys Arg Arg Leu 260 265 270 Ala Lys Gln Ala Ile Ala Asp Val Tyr Asn Ala Gln Glu Gly Met Asp 275 280 285 Asp Glu Ser Phe Glu Asp Ala Ala Glu Ile Val Gly Ile Leu Gly Lys 290 295 300 Met Val Lys Arg Gly Arg Asn Thr Ser Ser Ala Gly Asn Ser Thr Thr 305 310 315 320 Ala Val Lys Thr Pro Gln Glu Glu Arg Ser Glu Gly Ser Arg Thr Pro 325 330 335 Ser Ser Gln Thr Thr Leu Lys Arg Pro Ala Arg Val Pro Lys Ser Thr 340 345 350 Ser Ser Pro Ala Arg Ala Thr Lys Pro Thr Thr Ser Glu Gly Met Ser 355 360 365 Pro Ala Val Pro Asp Pro Thr Met Met Asn Pro Ile 370 375 380 <210> SEQ ID NO 34 <211> LENGTH: 592 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 34 Met Arg Asn Leu Glu Cys Val Tyr Thr Lys Ser Arg Arg Tyr Pro Ser 1 5 10 15 Ser Ile Phe Pro Thr Leu Arg Arg Ser Ser Thr Ser Pro Asn Thr Ala 20 25 30 Ser Ser Pro Asp Thr Asn Ala Ser Ser Ser Pro Cys Ser Asp Ser Ser 35 40 45 Gly Thr His Ala Gly Ile Ser Ile Pro His Ser Asp Ser Pro Ala Ser 50 55 60 Val Asp Glu Cys Gly Asn Asn Gly Leu Phe Asp Gly Glu Ser Leu Thr 65 70 75 80 Gln Leu Leu Val Ser Gly Ser Lys Ala Ile Ser Gly Gln Pro His Ile 85 90 95 Ala Ile Lys Pro His Val Leu Ser Thr Tyr Pro Leu Asp Asn Leu Leu 100 105 110 Ser Asp Ile Glu Gly Leu Leu Glu Gln Pro Val Ser His Leu Ala Gln 115 120 125 Ser Lys Glu Asp Leu Thr Lys Pro Asp Arg Met Phe Arg Thr Arg Leu 130 135 140 Pro Ala Val Ser Gly Glu Asp Asn Glu Tyr Leu Leu Arg Lys Gly Ala 145 150 155 160 Leu Leu Leu Leu Pro Pro Ala Leu Arg Asn Glu Leu Leu Ala Ala Tyr 165 170 175 Val Asn Tyr Val His Pro Leu Leu Pro Ile Leu Asp Leu Gly Asp Phe 180 185 190 Leu Ser Gln Ile Ile Val Gly Asp Asp Ala Gln Phe Lys Ser Pro Leu 195 200 205 Leu Tyr Gln Ala Val Met Phe Ala Gly Ser Ile Phe Ala Gln Arg Asp 210 215 220 Gly Thr Asp Glu Pro Glu Gln Leu Thr Arg Ala Leu Phe Glu Arg Thr 225 230 235 240 Lys Val Leu Tyr Glu Phe Glu Ser Glu Ser Cys Ala Tyr Thr Gln Ile 245 250 255 Gln Ala Leu Leu Leu Met Thr Leu Trp His Gly Asp Met Ser Arg His 260 265 270 Lys Gly Pro Ser Tyr Trp Leu Asp Val Ala Phe Ser Thr Ala Glu Arg 275 280 285 Ile Gly Leu Leu His Asp Glu Asp Trp Pro Cys Asp Ser Arg Ser Phe 290 295 300 Arg Ser Ala Met Trp Cys Cys Leu Tyr Val Arg Asp Arg Thr Ile Ser 305 310 315 320 Leu Gly Ser Arg Arg Pro Pro Arg Met Pro Val Asn Lys Tyr Pro Asp 325 330 335 Ser Ile Leu His Leu Met Ser Asp Ala Pro Arg Glu Tyr Asp Glu Ile 340 345 350 Ile Leu Glu Ser Leu Gly Ser Ala Ser Leu Met Leu Thr Ser Thr Tyr 355 360 365 Gln Glu Gln Ser Thr Met Leu Phe Lys Glu Leu Val Lys Leu Ser Tyr 370 375 380 Cys Val Gly Ala Ile Leu Asp Tyr Leu Tyr Glu Gly Ala Trp Val Arg 385 390 395 400 Ile Pro Thr Arg Arg Ser Ser Phe Tyr Ser Leu Ala Pro Lys Cys Ser 405 410 415 Ile Ser Pro Ser Ile Arg Pro Ser Cys Glu Lys Leu Leu Tyr Ser Trp 420 425 430 Ile Gln Phe Leu Pro Leu Asn Ala Val Tyr His Pro Pro Gln Leu Pro 435 440 445 Ala Asp Ala Glu Gly Glu Ser Ala Glu Thr Val Phe Leu Val His Lys 450 455 460 Ala Phe Leu Tyr Leu Leu Tyr Leu Ala Ser Met Ala Val Ile Tyr Arg 465 470 475 480 Thr Gly Thr His Ser Gln Gln Thr Ser Thr Ala Thr Pro Asn Met Glu 485 490 495 Gly Leu Arg Ala Ser Thr Ser Gln Ile Lys Lys Val Leu Ala Glu Leu 500 505 510 Gln Gly Met Gly Leu Leu Gln Phe Leu Pro Gly Ala Ser Val Thr Ile 515 520 525 Leu Met Phe Ala Val Glu Ala Ser Leu Leu Asp Leu Gln Gly Ser Asp 530 535 540 Thr Met Val Arg Arg Gln Ala Met Ser Asn Leu Tyr Ala Cys Glu Glu 545 550 555 560 Ala Ala Leu His Leu Met Gln Ala Tyr Pro Thr Ala Glu Met Ala Val 565 570 575 Leu Lys Thr Arg Thr Ala Arg Ala Asn Leu Leu Gly Leu Val Glu Thr 580 585 590 <210> SEQ ID NO 35 <211> LENGTH: 1076 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(35) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1062) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1037)..(1076) <400> SEQUENCE: 35 acttcactcg agataatgta ggcgacaaag tagccgggca tgcgaccgga ggactcggtc 60 agaggcacga atatgagagg ccagaacgcc gcacccatgt tccaagatgt tgtggcccag 120 taaaggttag gaaatggggt gttttgaacg tggaatcgtt cttccatcca gttgttgccg 180 gagccgtagg aaccggccgg gataccagtg aggatggaac tatacctatt agcactgcca 240 tcttcgagaa caaggctaag acatacatga aacaaacggt gaccgtgaga agccatttat 300 aagaggtgct ccaattgaac ctgaatatgt taaaactagt atactagcaa ctgcatgctt 360 gacgtacgga ttgtccgggt catccttgcc gttccaggta tggatctcaa ggtcctccct 420 gtcacgcaga tcattgtggc tgactacccc gacatggggg tcccggcgaa agaacgaccg 480 caccctgcaa cgtcacttag tggctgtctg acagggattg attggcaatg attggcagac 540 taaccgtccc agaaacgaat catcgaactc attggtgtgc actgtgctag cgcggcgcag 600 cgacgggcgt ggctcctcgg caggggtgga catgactttc tctgtgtaga ttccttgcaa 660 ggtatctgct tgacagatgc agagatgatg ttgagtaaaa tagggtgcag agaccctgca 720 gaacctgcat cggccggcgg cgacatgtcg tcatgaccga cccgggccga gcggaattaa 780 tgaaactcgg aacagccagg ggaatccgac gctttgagtg gccaatccat gtctatccac 840 atctgcattg aagtggatga aaggtggagg agggctggtt gccaaattct gttagggctt 900 ggaatgtaag caaccggttt cggtcatgac atcatcacag cgatgacttc atcactctcg 960 gacgagcata tatagttgtg cctgtcgtcg tatctcaaca aacatcaaca acaacaacaa 1020 tcatctactg catttaccaa actcatctct acctcaatca acttaattaa tgaagt 1076 <210> SEQ ID NO 36 <211> LENGTH: 1057 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(33) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1043) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1022)..(1057) <400> SEQUENCE: 36 acttcactcg agttctcaca agcaaatccg agaaacgcaa ttgaccatat gtccttagta 60 tgagggctag caggaatact tgtttgtcga tgcataccga ctaataaaaa tggttcttct 120 ttttcgcgca gggatttggt ttttcatttt tcatttttca tattttatgt tattttattt 180 tatttttttg ttttatttta ttttattttt tttttttaaa taagactggg tgttttcgtc 240 tcccacccct gctgaggaac tgtttgatat acagaagcct gagatacttg agattatgtg 300 acaaagaaat tgtaaacctg aatgtgacta aggcagaaga gagcaggtga ggccacacaa 360 tactgtacac ccacttccga tcgtcatcga aatgagcgtt gtgggtactg gaacgacaaa 420 ccatagatcg gatctgcatc cgaataggct ttactttgta cattggtagg agtttgaatg 480 tttgaagatc cggcagatga tcacctgctg cagggaataa tttgaaacct gtccgtctgc 540 tttgacccaa ttgacgaggt acgtgatttg atgctaccca agcagtatga atgcgacgga 600 ggtggctatc cttaccttgt catatgcaac tcataacggg tagtccccat ggagccacgg 660 ctatttattt ctaagggcca acagtgaaag attgtgagat cgtatacctc ctgggtatga 720 cgcaaccagg gtgagcactg gcattgctcc aatccatatg gtggaggtat gtctccgaga 780 ccatgacgca tgtagaccct gacctgtccc tgatccaggg atctggtttg tcacttggcg 840 tattttaatt aactgcgggt cagggtcttg ccgtccagac ccaatggatg aattgcctgc 900 gtggccctgc agttccagcg atcggcacgg cacggctatg acgtcggtgg gtgttccgtt 960 tgatatttaa ccatagaaga cagccttgtt atgctggttc ctgccgcaaa ataaaggtct 1020 atccttacca cgttaagtgt caaattaatt aagaagt 1057 <210> SEQ ID NO 37 <211> LENGTH: 1061 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(33) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1047) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1023)..(1061) <400> SEQUENCE: 37 atcctactcg agtggtgcct gtgaacgagg tcaccactga taaattgtct ggatccgtaa 60 cgtctgtatc agtggatggt actgaaaagt tccaaagatt gagggcagca tatgtagcac 120 atggtggtag ctctaagtgg ttcgaaactt cgatgaatgg gcgtgatata caggggggta 180 gcacctgtat agttgatgtc agcgaaggca cgggccatgc agtgatagac aagaaaagag 240 ggattgaata aaagactcga ttctgaaggg gagtaataca tctttcggct tctcaccacc 300 ccagatgtag gcgtgtgtaa ggtaagcaag cacaacatat gctctccgcc actcaggttc 360 ccccttcaac ttcttcgtag agagtatacc caggtcatcg acagacctcc tgatagtacc 420 agactgaatc aggccaggca agtcctgagc gatttcctcc cacggggaat agtaagggtc 480 ttccaatctc cgtacgggag gaacttccgg gaggaacccg ttctggaggg agacaccgta 540 cgcaacaagt gaaatgtctt gtgtataaag catgataata tatgcacttc agtagacaac 600 ttgatattca agcctccttg aaatgatcct catgcaggta agagtagatc caagtgttta 660 tattgtattc ccaaggaggg acggaagaaa accggatcca gagggctcga gatagggcca 720 cctgcatgag caaagacggg tcatttcccg tttcacaacg accattctca ggcagaaagg 780 cagtggatta gcggaaaact tagcaaccat ccggatttct ggagtggatg ctttgttatc 840 tcacttccgg caacacggat ggtgaggcgg aagcggaacg gtatccggat ccggagggag 900 ggacaaaccg aagtgccttg tgtcacacgc cggtccatat ataaatgtta attggtgacg 960 gtcagcggat tgtttctggt gttcatccat tttttcttga tatcctggac ccagttattg 1020 cactttaatt actatccaaa atccacatta attaatgaag t 1061 <210> SEQ ID NO 38 <211> LENGTH: 1097 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(34) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1083) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1060)..(1097) <400> SEQUENCE: 38 acttcactcg aggtccacac tctaattggg atgagcattg cctttcagga acttgacttt 60 ttgtggttgc atcttttgtt gccttaccca cgcaggcttc gtttatgaga tagcgatttc 120 aggcctcaaa ttgctgtcat cacttccggc cccaaactcg tgggcggtgc ccgcccatcg 180 ctggcaagaa gcacagacta acgccttact tcgcctagtt gcacacgatg catcaagact 240 tcctcctatt tcacaacgac cagctccttg ccatttgttg tttcgaatgc ccccttcccc 300 gtgccttgca tttctaattc ccatctatac ccttttgcgc tctcacgccc gcctgactat 360 caatttttct gccagcataa ccctgcgtgc agagcggttg acagcaggct tgctaagagt 420 cttcgtctag aagaactgtc tgggtggatt ccttttcgga ttaacttttg ctattttaaa 480 agctgattgc actacggagt acacccgcaa tctgattctc actgatactc caattaaagg 540 ccactaattg ctgacatttg taacatattt gcgtttgctt tccggttgaa attggcggct 600 ttgtgctttc gatcgctagc agtctctgtg catgtacaac tgatcggcta ttcagtccta 660 ttcggaagcg gcactgagag gcggagacaa gccacagata cgacgagtct gattttgaga 720 cgggcgcatc caacgaaagc ctgcgattgc aattgaagat ctcaattggg ggactgacaa 780 cctaatacct aaacatatcg aggattaggc gccaacacga gcatgggaag ctttggtgca 840 tgcgacggct gatccatttc taacaaccgt tttgcgcgcg cgtccgaggt aaggctcccg 900 atcccttggc gctttgcatg tttcactccg atgcatctac tgtttatatg agtgcggtgg 960 gcaagctgtg cccaccgact gagcattctc aaccggccca atgctgtggg atcgatcctc 1020 ccctccaaag ttgtcgctcg gttacacttg atcggagctt gttctgttat acaccaaacc 1080 atcttaatta ataggat 1097 <210> SEQ ID NO 39 <211> LENGTH: 1142 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(35) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1128) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1118)..(1142) <400> SEQUENCE: 39 atcctactcg aggcctttgg agaatgtaat atcccttgat caccccagac tcacatccag 60 attctgactg acctcgtgca tagccatatt taacttggtc cctgatgatc aaaacagtac 120 atggaactgg ggccgaacat ttccctagcc aaaggagcat cagggattca acgtgatcag 180 tacagccttg tagctcgctg aaactgccct ggagccaagc caaagcctag acagcaaggt 240 gcatgggcga gctttgcgag gatttagttg ttatttagta tgccaagatt aagccacgca 300 gaatggctta tggccatagt agcaacaacg gccacggaac gttcttccat gtggtatggg 360 gaaagccgcc ggatatttgg ggagggcgtg attgttccgg tggctatctt tccggtggag 420 ataacggcgg tggaccatca aaactcctca gcccggattg ctcggactcc gcatcgtcaa 480 gtatcggtttc atgacagcc attcgactac ttagaagaca caaatagcta tccaacttac 540 ccggacctttg ataataatt ttcagcagat ctgtcgatct cagagacaat aatgccaccg 600 ttctgtgtgga aggcactgc gccgtggttt aacaataata aaactccaaa ttgcccaaaa 660 gctagagcgaa ggaaagtcg agaagcccag tctagactgg cgggctgatc gtccggttca 720 caacctgattg acaaatcat ctgtgaccct cgatgttccg cggcgttgag cgggtgagaa 780 tgtggatccta gtggggaag acccaattgc cgccggaaaa ggtgagcttg tgctgcacat 840 gtccggtggct caattcagg gccatgccag gacattgctc ttcaaaggga tgcgattgct 900 attccctattt acttcttta acccgctgac gggcctgtct ggaccgagaa ttcttggtca 960 agtttcagtgg agcgatcca ggaaagaaca tccgatatgg acagttgccc tcgatctcag 1020 gtctgtagtcg tgtgacaca gaggcctcta ctcgcttgcc ttgcatgtcg gagctcaaag 1080 gtgcgatgtga attaacacg aggagcgact gtctgtcgca gatgcaactt aattaatagg 1140 at 1142 <210> SEQ ID NO 40 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 40 acttcattaa ttaagttgat tgaggtagag atgagtttg 39 <210> SEQ ID NO 41 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 41 acttcactcg agataatgta ggcgacaaag tagcc 35 <210> SEQ ID NO 42 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 42 acttcattaa ttaatttgac acttaacgtg gtaagg 36 <210> SEQ ID NO 43 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 43 acttcactcg agttctcaca agcaaatccg aga 33 <210> SEQ ID NO 44 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 44 atcctactcg agtggtgcct gtgaacgagg tca 33 <210> SEQ ID NO 45 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 45 acttcattaa ttaatgtgga ttttggatag taattaaag 39 <210> SEQ ID NO 46 <211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 46 atcctattaa ttaagatggt ttggtgtata acagaaca 38 <210> SEQ ID NO 47 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 47 acttcactcg aggtccacac tctaattggg atga 34 <210> SEQ ID NO 48 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 48 atcctattaa ttaagttgca tctgcgacag acagt 35 <210> SEQ ID NO 49 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 49 acttcactcg aggcctttgg agaatgtaat atccc 35 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 50 caacacctac ggcgtcaagg 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 51 taacccgggg agatgctgtt 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 52 cgatccggaa attcctcctg 20 <210> SEQ ID NO 53 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 53 gccgccgact cagcagtat 19 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 54 gtgcatgaac aacacggaga 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 55 actcagttgt gcgcctctcg 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 56 gtcgaactcg gcgtttctcc 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 57 ccggtgagga ggtgcttttc 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 58 agcatctcgc catccatcag 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 59 gccttcggac catggtatcc 20 <210> SEQ ID NO 60 <400> SEQUENCE: 60 000 <210> SEQ ID NO 61 <211> LENGTH: 1126 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: fusion gene <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(22) <220> FEATURE: <221> NAME/KEY: gene <222> LOCATION: (23)..(739) <223> OTHER INFORMATION: GFP <220> FEATURE: <221> NAME/KEY: gene <222> LOCATION: (740)..(1212) <223> OTHER INFORMATION: BLE <400> SEQUENCE: 61 gtcctgttaa ttaaccttca ccatggtgag caagggcgag gagctgttca ccggggtggt 60 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga 120 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa 180 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag 240 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta 300 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt 360 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga 420 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat 480 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga 540 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc 600 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa 660 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg 720 catggacgag ctgtacaagg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 780 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 840 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 900 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 960 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 1020 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 1080 cgtgcacttc gtggccgagg agcaggacta aaggcgcgcc tgaagt 1126 <210> SEQ ID NO 62 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 62 gtcctgttaa ttaaccttca ccatggtgag caagggc 37 <210> SEQ ID NO 63 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 63 gcactggtca acttggcctt gtacagctcg tccatg 36 <210> SEQ ID NO 64 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 64 catggacgag ctgtacaagg ccaagttgac cagtgc 36 <210> SEQ ID NO 65 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 65 acttcaggcg cgcctttagt cctgctcctc ggcca 35

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 65 <210> SEQ ID NO 1 <211> LENGTH: 168 <212> TYPE: DNA <213> ORGNISM: Bacillus subtilis <400> SEQUENCE: 1 cgactcagtc ctttcatata caatatgaag tgtaccgttt tccgcacttt ttcacaattt 60 cccataatct tttcattttt atcccacagt ttttgtttat gataaactca agtcataaac 120 ctatcaatat aaatagacat gtgaaaatag agaaacggag tgaacatg 168 <210> SEQ ID NO 2 <211> LENGTH: 277 <212> TYPE: DNA <213> ORGANISM: Bacillus subtilis <400> SEQUENCE: 2 ggctcttcac atcctttcaa cgtcattata aactagtttt aacatacggc aggcaatttt 60 cataatttca catattcttt tcatttttat cccacaatgt ttgtttatga tatggttaag 120 ggaagaaagg aagagaaaaa gagcgaggaa gatgtaggat gatcaaacat attctatttt 180 ggtgctttgc ttttctcctc attattggga caattgaact tgtacatgcg atgaatatgt 240 aacgataaat gaataaccgt taaaggagtg taagaac 277 <210> SEQ ID NO 3 <211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 3 gtgagcggat aacaatttca cacagg 26 <210> SEQ ID NO 4 <211> LENGTH: 70 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 4 gtggctcagc ttttttaagg aagggaggct ctcacctccc ttccctttat ttgtagagct 60 catccatgcc 70 <210> SEQ ID NO 5 <211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 5 cgtgaggtac cggcttctgt ttctgcc 27 <210> SEQ ID NO 6 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 6 catcacgaag cttatccatc atgttcactc cg 32 <210> SEQ ID NO 7 <211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 7 gaccggtacc tcaggatctt tcgcc 25 <210> SEQ ID NO 8 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 8 ggccatcaag cttataatcc atgttcttac actcc 35 <210> SEQ ID NO 9 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 9 gaaagattgt ttcagaagc 19 <210> SEQ ID NO 10 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 10 acagcgttgg gatccaagcc cttccatttt ggacatttgg 40 <210> SEQ ID NO 11 <211> LENGTH: 40 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 11 gggcttggat cccaacgctg tcgacgttgt aaaacgacgg 40 <210> SEQ ID NO 12 <211> LENGTH: 42 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 12 cgcatagctt tccggtcgcc gcagctatga ccatgattac gc 42 <210> SEQ ID NO 13 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 13 gatgattcgt ctttttgtag tg 22 <210> SEQ ID NO 14 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 14 cgatcaacat atgtgttcac g 21 <210> SEQ ID NO 15 <211> LENGTH: 1050 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 15 ataatgtagg cgacaaagta gccgggcatg cgaccggagg actcggtcag aggcacgaat 60 atgagaggcc agaacgccgc acccatgttc caagatgttg tggcccagta aaggttagga 120 aatggggtgt tttgaacgtg gaatcgttct tccatccagt tgttgccgga gccgtaggaa 180 ccggccggga taccagtgag gatggaacta tacctattag cactgccatc ttcgagaaca 240 aggctaagac atacatgaaa caaacggtga ccgtgagaag ccatttataa gaggtgctcc 300 aattgaacct gaatatgtta aaactagtat actagcaact gcatgcttga cgtacggatt 360 gtccgggtca tccttgccgt tccaggtatg gatctcaagg tcctccctgt cacgcagatc 420 attgtggctg actaccccga catgggggtc ccggcgaaag aacgaccgca ccctgcaacg 480 tcacttagtg gctgtctgac agggattgat tggcaatgat tggcagacta accgtcccag 540 aaacgaatca tcgaactcat tggtgtgcac tgtgctagcg cggcgcagcg acgggcgtgg 600 ctcctcggca ggggtggaca tgactttctc tgtgtagatt ccttgcaagg tatctgcttg 660 acagatgcag agatgatgtt gagtaaaata gggtgcagag accctgcaga acctgcatcg 720 gccggcggcg acatgtcgtc atgaccgacc cgggccgagc ggaattaatg aaactcggaa 780 cagccagggg aatccgacgc tttgagtggc caatccatgt ctatccacat ctgcattgaa 840 gtggatgaaa ggtggaggag ggctggttgc caaattctgt tagggcttgg aatgtaagca 900 accggtttcg gtcatgacat catcacagcg atgacttcat cactctcgga cgagcatata 960 tagttgtgcc tgtcgtcgta tctcaacaaa catcaacaac aacaacaatc atctactgca 1020 tttaccaaac tcatctctac ctcaatcaac 1050 <210> SEQ ID NO 16 <211> LENGTH: 1032 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 16 ttctcacaag caaatccgag aaacgcaatt gaccatatgt ccttagtatg agggctagca 60 ggaatacttg tttgtcgatg cataccgact aataaaaatg gttcttcttt ttcgcgcagg 120 gatttggttt ttcatttttc atttttcata ttttatgtta ttttatttta tttttttgtt 180 ttattttatt ttattttttt tttttaaata agactgggtg ttttcgtctc ccacccctgc 240 tgaggaactg tttgatatac agaagcctga gatacttgag attatgtgac aaagaaattg 300 taaacctgaa tgtgactaag gcagaagaga gcaggtgagg ccacacaata ctgtacaccc 360 acttccgatc gtcatcgaaa tgagcgttgt gggtactgga acgacaaacc atagatcgga 420 tctgcatccg aataggcttt actttgtaca ttggtaggag tttgaatgtt tgaagatccg 480

gcagatgatc acctgctgca gggaataatt tgaaacctgt ccgtctgctt tgacccaatt 540 gacgaggtac gtgatttgat gctacccaag cagtatgaat gcgacggagg tggctatcct 600 taccttgtca tatgcaactc ataacgggta gtccccatgg agccacggct atttatttct 660 aagggccaac agtgaaagat tgtgagatcg tatacctcct gggtatgacg caaccagggt 720 gagcactggc attgctccaa tccatatggt ggaggtatgt ctccgagacc atgacgcatg 780 tagaccctga cctgtccctg atccagggat ctggtttgtc acttggcgta ttttaattaa 840 ctgcgggtca gggtcttgcc gtccagaccc aatggatgaa ttgcctgcgt ggccctgcag 900 ttccagcgat cggcacggca cggctatgac gtcggtgggt gttccgtttg atatttaacc 960 atagaagaca gccttgttat gctggttcct gccgcaaaat aaaggtctat ccttaccacg 1020 ttaagtgtca aa 1032 <210> SEQ ID NO 17 <211> LENGTH: 1035 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 17 tggtgcctgt gaacgaggtc accactgata aattgtctgg atccgtaacg tctgtatcag 60 tggatggtac tgaaaagttc caaagattga gggcagcata tgtagcacat ggtggtagct 120 ctaagtggtt cgaaacttcg atgaatgggc gtgatataca ggggggtagc acctgtatag 180 ttgatgtcag cgaaggcacg ggccatgcag tgatagacaa gaaaagaggg attgaataaa 240 agactcgatt ctgaagggga gtaatacatc tttcggcttc tcaccacccc agatgtaggc 300 gtgtgtaagg taagcaagca caacatatgc tctccgccac tcaggttccc ccttcaactt 360 cttcgtagag agtataccca ggtcatcgac agacctcctg atagtaccag actgaatcag 420 gccaggcaag tcctgagcga tttcctccca cggggaatag taagggtctt ccaatctccg 480 tacgggagga acttccggga ggaacccgtt ctggagggag acaccgtacg caacaagtga 540 aatgtcttgt gtataaagca tgataatata tgcacttcag tagacaactt gatattcaag 600 cctccttgaa atgatcctca tgcaggtaag agtagatcca agtgtttata ttgtattccc 660 aaggagggac ggaagaaaac cggatccaga gggctcgaga tagggccacc tgcatgagca 720 aagacgggtc atttcccgtt tcacaacgac cattctcagg cagaaaggca gtggattagc 780 ggaaaactta gcaaccatcc ggatttctgg agtggatgct ttgttatctc acttccggca 840 acacggatgg tgaggcggaa gcggaacggt atccggatcc ggagggaggg acaaaccgaa 900 gtgccttgtg tcacacgccg gtccatatat aaatgttaat tggtgacggt cagcggattg 960 tttctggtgt tcatccattt tttcttgata tcctggaccc agttattgca ctttaattac 1020 tatccaaaat ccaca 1035 <210> SEQ ID NO 18 <211> LENGTH: 1071 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 18 gtccacactc taattgggat gagcattgcc tttcaggaac ttgacttttt gtggttgcat 60 cttttgttgc cttacccacg caggcttcgt ttatgagata gcgatttcag gcctcaaatt 120 gctgtcatca cttccggccc caaactcgtg ggcggtgccc gcccatcgct ggcaagaagc 180 acagactaac gccttacttc gcctagttgc acacgatgca tcaagacttc ctcctatttc 240 acaacgacca gctccttgcc atttgttgtt tcgaatgccc ccttccccgt gccttgcatt 300 tctaattccc atctataccc ttttgcgctc tcacgcccgc ctgactatca atttttctgc 360 cagcataacc ctgcgtgcag agcggttgac agcaggcttg ctaagagtct tcgtctagaa 420 gaactgtctg ggtggattcc ttttcggatt aacttttgct attttaaaag ctgattgcac 480 tacggagtac acccgcaatc tgattctcac tgatactcca attaaaggcc actaattgct 540 gacatttgta acatatttgc gtttgctttc cggttgaaat tggcggcttt gtgctttcga 600 tcgctagcag tctctgtgca tgtacaactg atcggctatt cagtcctatt cggaagcggc 660 actgagaggc ggagacaagc cacagatacg acgagtctga ttttgagacg ggcgcatcca 720 acgaaagcct gcgattgcaa ttgaagatct caattggggg actgacaacc taatacctaa 780 acatatcgag gattaggcgc caacacgagc atgggaagct ttggtgcatg cgacggctga 840 tccatttcta acaaccgttt tgcgcgcgcg tccgaggtaa ggctcccgat cccttggcgc 900 tttgcatgtt tcactccgat gcatctactg tttatatgag tgcggtgggc aagctgtgcc 960 caccgactga gcattctcaa ccggcccaat gctgtgggat cgatcctccc ctccaaagtt 1020 gtcgctcggt tacacttgat cggagcttgt tctgttatac accaaaccat c 1071 <210> SEQ ID NO 19 <211> LENGTH: 1116 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 19 gcctttggag aatgtaatat cccttgatca ccccagactc acatccagat tctgactgac 60 ctcgtgcata gccatattta acttggtccc tgatgatcaa aacagtacat ggaactgggg 120 ccgaacattt ccctagccaa aggagcatca gggattcaac gtgatcagta cagccttgta 180 gctcgctgaa actgccctgg agccaagcca aagcctagac agcaaggtgc atgggcgagc 240 tttgcgagga tttagttgtt atttagtatg ccaagattaa gccacgcaga atggcttatg 300 gccatagtag caacaacggc cacggaacgt tcttccatgt ggtatgggga aagccgccgg 360 atatttgggg agggcgtgat tgttccggtg gctatctttc cggtggagat aacggcggtg 420 gaccatcaaa actcctcagc ccggattgct cggactccgc atcgtcaagt atcggtttca 480 tgacagccat tcgactactt agaagacaca aatagctatc caacttaccc ggacctttga 540 taataatttt cagcagatct gtcgatctca gagacaataa tgccaccgtt ctgtgtggaa 600 ggcactgcgc cgtggtttaa caataataaa actccaaatt gcccaaaagc tagagcgaag 660 gaaagtcgag aagcccagtc tagactggcg ggctgatcgt ccggttcaca acctgattga 720 caaatcatct gtgaccctcg atgttccgcg gcgttgagcg ggtgagaatg tggatcctag 780 tggggaagac ccaattgccg ccggaaaagg tgagcttgtg ctgcacatgt ccggtggctc 840 aattcagggc catgccagga cattgctctt caaagggatg cgattgctat tccctattta 900 cttctttaac ccgctgacgg gcctgtctgg accgagaatt cttggtcaag tttcagtgga 960 gcgatccagg aaagaacatc cgatatggac agttgccctc gatctcaggt ctgtagtcgt 1020 gtgacacaga ggcctctact cgcttgcctt gcatgtcgga gctcaaaggt gcgatgtgaa 1080 ttaacacgag gagcgactgt ctgtcgcaga tgcaac 1116 <210> SEQ ID NO 20 <211> LENGTH: 3191 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 20 ggagttacgt cccagagatg ctgcggacaa tgccgatgcg gcatacttct catacgcatc 60 cgtcaagtaa ttcacaacag ccatgtagat actgtagatg ccaataccgg tagccccgat 120 gccgagggtc gggacaatcc aaggcacaga aggaaaactg gcccaaccat accagaacag 180 cccgcctgcg aacagcaagc tgccaatgat actagtatac agacgggcct ccgggatggg 240 tttcccgggc ttctctttgt tgcgtttcgc cgaccgcaaa tacagcatat cctgaagagg 300 attgacgaga gtaccgatta gagcacccac ggagatggcc aattgaatgc acccagtttg 360 caaagtattc atgccgtagt tggtggagaa ggtctgaacg acactgctga agaagagata 420 caggataccc caggcgaaag agatccagag ggtgaagaag gcaacgacgg gctcggtgag 480 gagcattcgt gtgggccgtt cgaaggagat ctgcatcaga cgccagacgc tggtgttgtc 540 tagctcggct tcggcgtaga tggggcgctg cttttctttg cggagcttct tggcccgacg 600 ggtgaggatc acgtccgcgc gggtttcgta caggatgaac cagaagatgg gtaggagccc 660 tgttagatag ataatttgga tgtagaagat ccagcgccac ggggcggttt tgtggatggc 720 tacgatggcg gatccgatga atgggcccag ggcaattcca accacactgg taaaaccgaa 780 gagcgacata gggagactcc tcgctttgtc accataccag acatcactga tgctaccgcc 840 gacgatgttg atcgatacgg aggacgcacc accaccgaag aaacgcgtca ctacgagagt 900 agcgtagttt tgggcgaagg cggaggggaa tagcgagatg atgagaataa tgtaggcgac 960 aaagtagccg ggcatgcgac cggaggactc ggtcagaggc acgaatatga gaggccagaa 1020 cgccgcaccc atgttccaag atgttgtggc ccagtaaagg ttaggaaatg gggtgttttg 1080 aacgtggaat cgttcttcca tccagttgtt gccggagccg taggaaccgg ccgggatacc 1140 agtgaggatg gaactatacc tattagcact gccatcttcg agaacaaggc taagacatac 1200 atgaaacaaa cggtgaccgt gagaagccat ttataagagg tgctccaatt gaacctgaat 1260 atgttaaaac tagtatacta gcaactgcat gcttgacgta cggattgtcc gggtcatcct 1320 tgccgttcca ggtatggatc tcaaggtcct ccctgtcacg cagatcattg tggctgacta 1380 ccccgacatg ggggtcccgg cgaaagaacg accgcaccct gcaacgtcac ttagtggctg 1440 tctgacaggg attgattggc aatgattggc agactaaccg tcccagaaac gaatcatcga 1500 actcattggt gtgcactgtg ctagcgcggc gcagcgacgg gcgtggctcc tcggcagggg 1560 tggacatgac tttctctgtg tagattcctt gcaaggtatc tgcttgacag atgcagagat 1620 gatgttgagt aaaatagggt gcagagaccc tgcagaacct gcatcggccg gcggcgacat 1680 gtcgtcatga ccgacccggg ccgagcggaa ttaatgaaac tcggaacagc caggggaatc 1740 cgacgctttg agtggccaat ccatgtctat ccacatctgc attgaagtgg atgaaaggtg 1800 gaggagggct ggttgccaaa ttctgttagg gcttggaatg taagcaaccg gtttcggtca 1860 tgacatcatc acagcgatga cttcatcact ctcggacgag catatatagt tgtgcctgtc 1920 gtcgtatctc aacaaacatc aacaacaaca acaatcatct actgcattta ccaaactcat 1980 ctctacctca atcaacatgc ctatctccat tccctccgcc tccagcgtcc acgacctctt 2040 cagcctgaag ggcaaggtcg tcgttatcac cggcgcttcc ggccctcgcg gcatgggcat 2100 tgaagccgcg cgtggctgcg ccgaaatggg cgccaacatc gccctcacct actcctctcg 2160 tcctcagggt ggtgagaaga acgccgaaga acttcgcaac acctacggcg tcaaggccaa 2220 ggcctaccag tgcaacgtgg gcgactggaa cagcgtcaag aagctcgtgg acgacgtgct 2280 ggccgagttt ggccagattg acgccttcat tgccaacgct ggcaagacag ccagcagtgg 2340 tatcctggat ggttccgttg aggactggga agaggtcatc cagacagacc tgacgggtac 2400 cttccactgc gccaaggcgg tgggcccgca cttcaagcag cgcggaacgg gcagcttcat 2460 catcacctcc agcatgtcgg gtcacattgc caacttcccg caggagcaga cgtcgtacaa 2520 cgttgccaag gcaggctgca tccatatggc ccggtcattg gccaacgagt ggagggactt 2580 tgcccgcgtg aacagcatct ccccgggtta catcgacacg gggctgtcgg actttgtgga 2640

taagaagact caggatctgt ggatgtcgat gatccccatg ggacgcaacg gtgatgccaa 2700 ggagctgaag ggtgcatatg tctatctcgc cagtgatgcc agcacataca cgacgggtgc 2760 cgatttggtc attgacggag gatacaccgt gcggtaaatt atcatccgcg gtcggataga 2820 aaataatgtt tatgacatgt atgtaatgaa tatgaatgtt caacaatagt cttcaattct 2880 tcacttgcta gtatcctgag tatcttgtcg tatagctcaa caacctggca tggccgggcg 2940 cgcagtgtag ctctcccaag tctatggata caacttgcgt cagtgtggat ctttggctca 3000 gattcggagg ggcagtgggg taaacaagaa cacgcagcca ctctcggctt tgcggatccc 3060 cacggaacgc cagtcaaccc tgaagattca gtccgggcct gggctcggac tgatttggtg 3120 aggggattct gcatccgtcc aaaccttatc atcgttggtg agacggactc cggacagcca 3180 ggagccttat t 3191 <210> SEQ ID NO 21 <211> LENGTH: 4031 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 21 ggggtcaaag gacacaatgt cgaaatagcg agattgctgt gcatcatcgg ggctgtctac 60 agtccgctgg atttctttcc ctcggttaag tatttcctgc acgccggacc cgccagggcc 120 acctgtattc ctagtaagcg accgtggtgg ggtgacagaa tggtggcggt ggtggcattg 180 ctgcctatac ctgggttcac gattatggag ccaccgtaac gctcgtcggc aggatctacc 240 tgtgcaggca gttttatgac ggccagggca gccgttttat tttcaatggc actagctgac 300 cagtctaggg gaacttcgag acgtgcacat agatagtcct ggtagcaggg gatgaaattt 360 agtgctgagc ctggcgcagt ctggtaggag cgggttagac tgtgagaaat tataataggg 420 cggatgagat cagtacgcta ctccaggaga agtccccggc ttgcaccgca gacggcatca 480 tgggttggtg tcgcagggag gagggtgagt tgagtaagag agaattaatg aaaaggatgg 540 agtagccgac catataagta ctggatgaag tgagcgagtt agcgtcagcc acgacgggct 600 gacatgcatg atcggggcac cgccggcaac cgatacagcc tcagctcgac gggaaaacgt 660 ggttacctgg acttagaaga tgataataca atgaaaagat agcgtagatt gccataaata 720 cgccacaatc tatttattag gcaacaatca agtaagagca gatttggcga ctcatagtag 780 tccccatgga ggggcgcgat ggttttcttc gccgaaataa tcgcttaatg cctctaacga 840 agatctcaga acctccaaac tgtagtattc ttctcacaag caaatccgag aaacgcaatt 900 gaccatatgt ccttagtatg agggctagca ggaatacttg tttgtcgatg cataccgact 960 aataaaaatg gttcttcttt ttcgcgcagg gatttggttt ttcatttttc atttttcata 1020 ttttatgtta ttttatttta tttttttgtt ttattttatt ttattttttt tttttaaata 1080 agactgggtg ttttcgtctc ccacccctgc tgaggaactg tttgatatac agaagcctga 1140 gatacttgag attatgtgac aaagaaattg taaacctgaa tgtgactaag gcagaagaga 1200 gcaggtgagg ccacacaata ctgtacaccc acttccgatc gtcatcgaaa tgagcgttgt 1260 gggtactgga acgacaaacc atagatcgga tctgcatccg aataggcttt actttgtaca 1320 ttggtaggag tttgaatgtt tgaagatccg gcagatgatc acctgctgca gggaataatt 1380 tgaaacctgt ccgtctgctt tgacccaatt gacgaggtac gtgatttgat gctacccaag 1440 cagtatgaat gcgacggagg tggctatcct taccttgtca tatgcaactc ataacgggta 1500 gtccccatgg agccacggct atttatttct aagggccaac agtgaaagat tgtgagatcg 1560 tatacctcct gggtatgacg caaccagggt gagcactggc attgctccaa tccatatggt 1620 ggaggtatgt ctccgagacc atgacgcatg tagaccctga cctgtccctg atccagggat 1680 ctggtttgtc acttggcgta ttttaattaa ctgcgggtca gggtcttgcc gtccagaccc 1740 aatggatgaa ttgcctgcgt ggccctgcag ttccagcgat cggcacggca cggctatgac 1800 gtcggtgggt gttccgtttg atatttaacc atagaagaca gccttgttat gctggttcct 1860 gccgcaaaat aaaggtctat ccttaccacg ttaagtgtca aaatgagtgg ggtaagcttt 1920 gcccgtcagg ccttactcca ggctgctctt ctttcgattg tctccgcaat cccggcgccc 1980 acggccttcg tcaacaatgt cgctccgccc gagcccatca tcaccccttc gccggttcaa 2040 catcgacctt ctcgagtcgc tggtcgaaac attctgagtg acgtcgattc tgatattaac 2100 agcatccttt ctggccttgg atccgaccta ccctcctggg tcgcatcggg tgtgcccaac 2160 tacttccagg gtttccctac tggggatgca gtggtgagct ccttgggctt gaacagcgct 2220 gagttggcag ccttgcccac caatgtcttg aatatcgacc catatgccaa ctggactagc 2280 tctggctgga acgttcgctt ccacggaaac gtctacaagc agcccaacac ctccatctcc 2340 gacctcaatg atttggctga tgttttcctc ggcaatacaa gcatctccga cctttccgaa 2400 tccgagcaga aacaggctcg gaatttaaca gcggaaatat tagtggtcca gcaagctcac 2460 gtcgccgtaa acacaatcca cctggagcct gcaccgagtc agggatccag cggacagtca 2520 ggcggcggtg gatcctctaa taccactggc ggcacccaag acctcactct gccctacaac 2580 actacagttg aaggcgattt tgacactttc gtaccgatta gcagcaatgg cttgacggca 2640 ggtaatgaaa cttcggcgat acaacggctt aacgtccatg tggagggcgc gacaatagga 2700 aacagtactg cctatctcgt acctccgacc gggctcactg tcgtttcgga catcgatgat 2760 atcctacgcg tcaccaaaat ctatgaacca gagcaaggtt tactcaactc atttgcgcgc 2820 cccttcaggc cgtgggagaa tatgccggac atctatcgca actggtcgat cagtctcccc 2880 aacctgcact ttcactacct agtaagtctg ctagatgtcc ctgattgcat tgttgctgca 2940 tactcaccac tacactacag acgacgaccc ccgaacaagt caccaggaac tacatgcaat 3000 tcatctacga caactacccc ggtggatctt tcgacacccg tcccctcaac tttagcgacg 3060 tttccgccac cttgtcgatc cggaaattcc tcctgcaaaa ggtcttcgag acctttccgc 3120 agcgcaagtt catcctcatc gccgacacca gcaacagcga cgtcatgcgt gactatcctg 3180 aaatggcgac cgacttccct ggtcaagtgc aatgtatttt tctccggaac accagcgcca 3240 ccgactcagg ggacaaattc ccatacgata cctctgggtt taagaagctc aatcagtcaa 3300 actacatgtt cttcttgcat cccgatgact tgacgaatct agacatcgcg aatggccagt 3360 gttacaacac atccattccg cagaatttaa cgtttagtta tcagggtttg ccattggggc 3420 tgggtgatga acccacagct gtcaatgggt ctgccaatca tactgctgag tcggcggccg 3480 gtttgtcggt aaagggtggg actgatatgc agggtttgat ctttctgttc accctggtta 3540 cggcctattc cattttcttg taaatatcag ttacgatgta tgagagataa cgactttgat 3600 atactcacgg ctcaatttac gttccaaaac cgaatacagc ggatatctat gctcgatgca 3660 aggactacca ctatgttaca ctactaaggg tttatctact atgggaaata ttagtgcttg 3720 gtttgttaca gctcactcga gcttgcgtta ctaaccactt ttatcggtcc atggatccac 3780 cgcccaacgc agttatccca ggaggtggcc gcaatggcca gtcaagtcaa ttaaatctcc 3840 tctgctagga attgcatggg ttttgacaga cagaactcgg ggaaagtatc gatattcaat 3900 gatctgacgg tggctccgaa attcgctagg ctagcccttc atcactagta atattagttg 3960 tccaactttc gcgctttaca ctttccatag caaaccccca attcccagcc aaatcctcat 4020 atcctctagg a 4031 <210> SEQ ID NO 22 <211> LENGTH: 3771 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 22 gcttctatgg tatcaagggt cgcgtagcca aaagtttaac tgcatcgctg cggtattagt 60 cggtgtagct ggctgcagct ttcgttgtgt ccctggtctg tttaagaaac ggtatcagat 120 ctgtcccgcc cgtccccctg acaccggcat tgagtttctc atgagaattg tccactattg 180 tcgacgttgt tgtggctagg tttgttctcg aagatacttg gtttggcggc ggagtccttg 240 cagccatgat gatgtatcga gtcaccatcc gcacatgctt atcgcgaaat cccccaagtg 300 acataaccgc catgttatat gcatccctca aagcagatcc gggcttgtga cttaatacat 360 aggatctcac attagacgtg cggagcatca tttccaagaa gcgtcggtga gggcctggca 420 tgtagtttcg catgttcttg aatatacagt cagtatcgac attgctttgt ctggataact 480 cctcgaagat cagtccttac ctgcataaaa ccagtcatga cttgaggatt cgacttgccg 540 tgaggcttgg tctccccagt ggccgaatgt tccacaccca agaaaatgtc gaaggcttgg 600 atcagagaac tttgagcatt gctaccacca ctgtattgat gccattcgcc tcgtccttcc 660 ccaacatcat agaatacgcc gtttggcagc ccggccgatg ccatgttctt gctgccggct 720 aggtatggac gtaattgatg gtaaaagacg ggcggcgcac acttttcata catgcgttcc 780 aagaggtcac cgagttcctt gagaccataa ttgagctgct ccagacgaga aataacacgc 840 tggctatcat caacattagc tgcatgcata gcatccagca ttagctcgat gagctgtgct 900 cccttggcct cgatagcgac ggagaggacg aaaaaccatt cttcgtcttt ggtgcctgtg 960 aacgaggtca ccactgataa attgtctgga tccgtaacgt ctgtatcagt ggatggtact 1020 gaaaagttcc aaagattgag ggcagcatat gtagcacatg gtggtagctc taagtggttc 1080 gaaacttcga tgaatgggcg tgatatacag gggggtagca cctgtatagt tgatgtcagc 1140 gaaggcacgg gccatgcagt gatagacaag aaaagaggga ttgaataaaa gactcgattc 1200 tgaaggggag taatacatct ttcggcttct caccacccca gatgtaggcg tgtgtaaggt 1260 aagcaagcac aacatatgct ctccgccact caggttcccc cttcaacttc ttcgtagaga 1320 gtatacccag gtcatcgaca gacctcctga tagtaccaga ctgaatcagg ccaggcaagt 1380 cctgagcgat ttcctcccac ggggaatagt aagggtcttc caatctccgt acgggaggaa 1440 cttccgggag gaacccgttc tggagggaga caccgtacgc aacaagtgaa atgtcttgtg 1500 tataaagcat gataatatat gcacttcagt agacaacttg atattcaagc ctccttgaaa 1560 tgatcctcat gcaggtaaga gtagatccaa gtgtttatat tgtattccca aggagggacg 1620 gaagaaaacc ggatccagag ggctcgagat agggccacct gcatgagcaa agacgggtca 1680 tttcccgttt cacaacgacc attctcaggc agaaaggcag tggattagcg gaaaacttag 1740 caaccatccg gatttctgga gtggatgctt tgttatctca cttccggcaa cacggatggt 1800 gaggcggaag cggaacggta tccggatccg gagggaggga caaaccgaag tgccttgtgt 1860 cacacgccgg tccatatata aatgttaatt ggtgacggtc agcggattgt ttctggtgtt 1920 catccatttt ttcttgatat cctggaccca gttattgcac tttaattact atccaaaatc 1980 cacaatgtcg aagtcgaatg gcgtcagcct ggcattcccc gccgaagcag cgacaaagga 2040 atatgcagca tctctagact cttctgatag acttgctgca tttcgggaga agttcatcgt 2100 cccatcgaaa gcaaatattg cttccaaaaa gctagcaaag cctggtgggt tccatcctgg 2160 caactgtaga cctcgtcact aatgtcacag gcctttcgcc cgagtcatgc atttacttct 2220 gcggtaactc acttggcatt caacccaagg ccacagcaaa gtacttagaa gcacagttgg 2280 acacttggtc atctattgga gttggcggcc acttcactga tcttgagggc tcacccttga 2340 agcaatggca gctgctctca gagcaagcag ctgattcaat gagcaagatt gtgggagcaa 2400

agccagaaga agtcgcagcg atgggaacac ttacaacgaa ccttcatctt ctgctggcta 2460 gtttctataa accgactcaa acaaagcaca agatcttgat ggattggaag gctttcccaa 2520 gtgatcacgt aagttcgaaa gaagctgcct tctgctatca cggcactgac gattatatag 2580 tacgctatcg aatctcatat tgcctggcat gacctggatc ctaaggagtc tatggtgctc 2640 atcggtccag atgaaggcga atacgagata tccacccaga agattttctc ttatatcgac 2700 aagcatgctg atgaggctgc tatgattctt cttcctggta ttcaatatta tactgggcaa 2760 ctgttcgata tccagaaaat tacaaaatat gcccattcgc gcaacatggt tgttggctgg 2820 gatctggccc atgcgttcgc taatgttgag ctaaagctgc atgattggaa cgttgatttt 2880 gcagcatggt gcacatataa gtacggaaac gccggtcctg gagcaatggg ggggcttttc 2940 gtgcatgaac aacacggaga ggtcgattac agcgcgggag aagatgcacc caaattccgc 3000 catcgtctga ccgggtggta tggtggcgat cgatcggtaa gattcaagat ggacaacagt 3060 atgtgtgcac ttggctatag aacaattttg tgggctagta agctgatacg attgataacc 3120 agagttcaaa cctattcctg gggcaggagg atttcagata tcaaatcctt cggccatcga 3180 ccttgcgtgt ctttgtgccg ctttatcggt gtttgatgag acgtcaatgg cagatcttcg 3240 cagaaaatca ttgaagctaa cagcatacct tgagttcctt ttgctcaggg attatgaaga 3300 agaatctagg ccattcagca ttatcacgcc caaggacccc gaggcgagag gcgcacaact 3360 gagtctattg ctcaagcctg gtctattaca gaatgttgca cagaagttgc aagaagcagg 3420 aatagtctgc gataaacggg agccgggtgt tgtgcgtgtc gctccggttc ctttgtacaa 3480 cagcttcagc gaggtttgga catttgtgaa gatcttcaag gatgcactgc agcagtgatg 3540 ggtctcacta gatcatttgg tcaggactga gaccagattt gtcaagttcg ctccgatgaa 3600 tgatttttta gatagatacc aaattatttg attcttagat ttgttgaagc tctcactgcg 3660 acatggtcca cagccactgt gcaaaagcaa ataactgaag taggaactct atgatagacc 3720 cgtactgttg aggttataga tttccaaagc cgttccttgg atcaccactt c 3771 <210> SEQ ID NO 23 <211> LENGTH: 3562 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 23 cctgtgcttg tggtatgaca gagccactga tatcatctcc tctcatctcc agttgtaatc 60 ggaattgatc gagggggcca atcttccaca cagccccatg gaagacgcac agatgctttc 120 gatgccacgc tggcagacgg ccgccaactg tcatcgtcca gtgctgccgc cattcccatg 180 caaaagccaa cgctaagaag cgcgcgggcg ccgagtttcc acgcttgcag cttagttcag 240 cgacccagct cagtggcggc tcgctcatgc atagaaagat gggtttggac ccgcgctggg 300 cctttcatct gcaacactgc tcgcagacca tgtgtgactt ttggctgcaa aaaaaaagag 360 gctggcgaca gctgatccac tgtgctattc ctatctcatc ctccccggct ggatcgaaag 420 ctgcacggca ggggaaatca acagttgggc tctgacacgt atcgagctaa ctgatgcgct 480 gtccttccgc ctaatgccta ctaagtctta gggacgggac catcatccaa catgccacct 540 tgtccttcgt aattcctttt tgatctcggt gatggtggcc acccaggaaa tgacgttcct 600 caccttggct gcaattggtg cccgatgtct catctccgct cagaacggcg gcgagggcag 660 aataaaaatt gacgggcaca gcaggaacta atcaaacata ccggcggtct tctaaatgat 720 cgtatctccc ggaatactcc ctctttctac agaagcgggg atccattcgc cctatatgta 780 tagtggagct ttgccgcact gattcgcacg gcattatacc tccgcactgc cgtgccttca 840 tccacttgat tgtggccagg gaaatatcaa cggggcagat aacttgccct tgcatcatta 900 ttgctccgca cattcattca ttatcgctct gatcccacat cctcacgcag ctgcgaccgg 960 agtgtccaca ctctaattgg gatgagcatt gcctttcagg aacttgactt tttgtggttg 1020 catcttttgt tgccttaccc acgcaggctt cgtttatgag atagcgattt caggcctcaa 1080 attgctgtca tcacttccgg ccccaaactc gtgggcggtg cccgcccatc gctggcaaga 1140 agcacagact aacgccttac ttcgcctagt tgcacacgat gcatcaagac ttcctcctat 1200 ttcacaacga ccagctcctt gccatttgtt gtttcgaatg cccccttccc cgtgccttgc 1260 atttctaatt cccatctata cccttttgcg ctctcacgcc cgcctgacta tcaatttttc 1320 tgccagcata accctgcgtg cagagcggtt gacagcaggc ttgctaagag tcttcgtcta 1380 gaagaactgt ctgggtggat tccttttcgg attaactttt gctattttaa aagctgattg 1440 cactacggag tacacccgca atctgattct cactgatact ccaattaaag gccactaatt 1500 gctgacattt gtaacatatt tgcgtttgct ttccggttga aattggcggc tttgtgcttt 1560 cgatcgctag cagtctctgt gcatgtacaa ctgatcggct attcagtcct attcggaagc 1620 ggcactgaga ggcggagaca agccacagat acgacgagtc tgattttgag acgggcgcat 1680 ccaacgaaag cctgcgattg caattgaaga tctcaattgg gggactgaca acctaatacc 1740 taaacatatc gaggattagg cgccaacacg agcatgggaa gctttggtgc atgcgacggc 1800 tgatccattt ctaacaaccg ttttgcgcgc gcgtccgagg taaggctccc gatcccttgg 1860 cgctttgcat gtttcactcc gatgcatcta ctgtttatat gagtgcggtg ggcaagctgt 1920 gcccaccgac tgagcattct caaccggccc aatgctgtgg gatcgatcct cccctccaaa 1980 gttgtcgctc ggttacactt gatcggagct tgttctgtta tacaccaaac catcatggat 2040 gtgactaatt tggctgtcga ttgtcccgtt atagagaccg gttcccttgc tttgtggatg 2100 gcttcctccg aagtggacca gaaggccttg ggcaactttg cggccataac gggcctcgag 2160 aacccgttcc tgtccgcgat gctgtacaag attctggggt tgtctgttat gttatcgaag 2220 aaactcctcc gtgcacgacg attgcggcgc ctagacccca cccgagagac taaatcgctt 2280 catctttatt accatatcat ctggctttcg cgtgagggtc ttctgatatt agaggagttc 2340 gtcctgccac tggttgaggg atttatggaa ctcaaaattt tagcttacaa gctgcgcgcc 2400 tccttttacc atatattcgt cctatttcag aaccagcctg ctgtccactc gccgggtatc 2460 gttagtctgc cgagtagtac acctttatcc aacggcgtga cagaagcgga gtcaccatcc 2520 aaggattcca atttgagatt ttcatttcag ctgaagcccg acacgataac ggtttccggg 2580 aagcccagtt ccgcctcgga tagtgcgcca cgaggcaagg ttacgcaggc acccccgggc 2640 tttgcgccgg ttcaaccgcc caagtcgaac tcggcgtttc tcctccctgc tctcgattat 2700 acccctacag ctaccgcatg cttcaatcat gccgccctct tggcagatca attcctcccg 2760 gggtcgcacc cgctccgtct gtccatcaag cttgaatttg ccgcctacct ctacgattgc 2820 ctacatgacg ctcatgcctg tcgacggttg gccaagcaag ccattgccga tgtgtacaat 2880 gcgcaagagg gcatggacga cgagagcttc gaggatgcag ctgagattgt gggtattttg 2940 ggcaagatgg tgaagcgggg aaggaacaca agcagtgctg gaaatagcac cacggctgtg 3000 aaaacgcccc aggaggagcg aagtgagggc agtcgaacac catcctctca gacaacgttg 3060 aaaaggccgg cgcgagtgcc gaaaagcacc tcctcaccgg cgcgcgcgac caagccgacg 3120 actagcgagg ggatgtcccc cgctgtgccc gaccccacca tgatgaatcc catatgatgc 3180 ctttccctac gacctgtata tgcttcttct tggaactatg gagtcgcgca tgagcgcttg 3240 gcctgtgact tctacttgac ttgcacctgt ctgtatcatc tcggtaacgg agttgcggcg 3300 atagactgga attgggcatc agcttgcggg gagctgtgga atgagcgatt aagcgcgaca 3360 aaaacttgag gtggaaggga cagaccagcc aatttgtgat ggtgactgta gatagataga 3420 tgtgtgcaat acaaagtatg ttgttgatgt catgcacccc acttgtgtat gcttgtctgg 3480 acaatggacc tacacttagt cgcaacaact gtatgtctca agccaatggc catggggcag 3540 gatggatcga tggatctctt gt 3562 <210> SEQ ID NO 24 <211> LENGTH: 4361 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 24 cgacggacta acgtcgcgcg ccccatttca cgtcttgaag acagatgtgt atgcgattgc 60 tcgcgaaatt ctcatccgat tccgtcggct tgacctgaca tctggaatag acgaacagga 120 cttggaagct atcattcgta ttaccgaaga gtccaacgta cttctcactg actggtggaa 180 gtccgtcccg aaactgtatg acttcgatta ctggaaagca gacggtcgat gggaaatgat 240 tgaaggccag ctgcaaaggc ttccctccga agcaaggcaa caggtggaga caatatatct 300 acaggctgct gttctccagt tgacgtacga cggcatggtc atccaagcaa atagaccact 360 gttggagcga agaatcaaca ggctaacatc ttcgcgggcg atcgtggatg cgatgcatag 420 ggcgcttgac cgagccactg ctgctgctct ccgcatatcc cgtgttccag ttcacaagct 480 gaaaaatcat tttgctatag cagtcgtatc gatgcaagag ttcactgctg gtgtgattct 540 ctgtatacct cccacggtga agcctttaac ggccaccgct catgaggcga aggacggggt 600 ggtaagaata atacgcgcca gtcgaagtct caagagccac gatcgaatag caaggcaaac 660 tgaacaactt ctgacggaac tactcaaagt taccacccaa cgcgagataa cgcacgcctt 720 ggccgataat ctcgacacca atttcccctc cgatacgcgg ctaacaggct ccgttgttgc 780 cggcgcctca agtgactcct tgggtgcagg acgctccagg ccagttcccg gatccgcagc 840 gcaggcagtt gtagatccta tccaaggagc ctgggactca gaattcaatg ccctccctgc 900 cgtaacggga ccttatgatt tcctccctgc tcaagccttt caacagctgg acgatacatt 960 tggtgccttt ggagaatgta atatcccttg atcaccccag actcacatcc agattctgac 1020 tgacctcgtg catagccata tttaacttgg tccctgatga tcaaaacagt acatggaact 1080 ggggccgaac atttccctag ccaaaggagc atcagggatt caacgtgatc agtacagcct 1140 tgtagctcgc tgaaactgcc ctggagccaa gccaaagcct agacagcaag gtgcatgggc 1200 gagctttgcg aggatttagt tgttatttag tatgccaaga ttaagccacg cagaatggct 1260 tatggccata gtagcaacaa cggccacgga acgttcttcc atgtggtatg gggaaagccg 1320 ccggatattt ggggagggcg tgattgttcc ggtggctatc tttccggtgg agataacggc 1380 ggtggaccat caaaactcct cagcccggat tgctcggact ccgcatcgtc aagtatcggt 1440 ttcatgacag ccattcgact acttagaaga cacaaatagc tatccaactt acccggacct 1500 ttgataataa ttttcagcag atctgtcgat ctcagagaca ataatgccac cgttctgtgt 1560 ggaaggcact gcgccgtggt ttaacaataa taaaactcca aattgcccaa aagctagagc 1620 gaaggaaagt cgagaagccc agtctagact ggcgggctga tcgtccggtt cacaacctga 1680 ttgacaaatc atctgtgacc ctcgatgttc cgcggcgttg agcgggtgag aatgtggatc 1740 ctagtgggga agacccaatt gccgccggaa aaggtgagct tgtgctgcac atgtccggtg 1800 gctcaattca gggccatgcc aggacattgc tcttcaaagg gatgcgattg ctattcccta 1860 tttacttctt taacccgctg acgggcctgt ctggaccgag aattcttggt caagtttcag 1920 tggagcgatc caggaaagaa catccgatat ggacagttgc cctcgatctc aggtctgtag 1980 tcgtgtgaca cagaggcctc tactcgcttg ccttgcatgt cggagctcaa aggtgcgatg 2040

tgaattaaca cgaggagcga ctgtctgtcg cagatgcaac atgagaaatt tggagtgtgt 2100 ctacacaaaa tcacgaaggt atccctcgtc tatctttccg tgtgtattat tagccgagag 2160 tcacgcaatg ctctttgtat acaatgaaat aatgttggag ctagagaatg atttctgata 2220 gttatatgtt acaggacact caggaggtcg tcaactagtc cgaacacggc gtcctctcct 2280 gacactaatg cctcatcttc gccatgctcg gactctagtg ggacccatgc tggcatttcc 2340 attccccact cagatagtcc cgccagtgtg gacgaatgtg ggaacaacgg ccttttcgac 2400 gggggtaggt aatatgtcat gcttacctgc gacactcact caccattctt ctagaatctc 2460 taactcaatt attggtctct gggagcaaag ccatatcagg ccaaccacat atcgccatta 2520 agccacatgt tctgtctacc tatccccttg acaacctcct ctcagacatt gagggactcc 2580 tcgagcagcc agtctctcat ttggctcaaa gcaaggaaga tctaacaaaa ccggaccgga 2640 tgtttcgaac tcgccttcca gcggtctcag gtgaggacaa tgagtatctg ctaaggaaag 2700 gagctctgct gctcttgcca ccagccttgc gcaacgaact attggcggca tatgtcaact 2760 acgtccatcc gctattaccg attcttgatc ttggagactt tctctcacaa atcattgttg 2820 gagatgatgc tcaattcaag tcaccactat tataccaagc tgtcatgttc gcgggcagta 2880 tttttgctca gcgagacggc acagacgaac ctgaacagct aacgagggct ttatttgaac 2940 gtacaaaggt tagtaatggt tacaattaga catatcttag atatactgag cgatttccag 3000 gttctatacg agtttgaatc cgaatcatgt gcctacactc aaattcaagc tctcttattg 3060 atgactctct ggcatggaga tatgtcacgc cacaaaggcc cgtcttactg gcttgacgtc 3120 gccttctcca ctgcggaaag aattggccta ctccatgatg aagactggcc atgcgatagc 3180 cgttccttcc gatccgctat gtggtgttgc ctttacgtac gtgatcgaac aatctcgctt 3240 gggtctcgtc ggcctccacg gatgccagtc aacaaatatc cggactcaat tctccatctt 3300 atgagcgacg cacctcggga atatgacgaa ataattcttg agagtctagg tagcgcttct 3360 ctgatgctta caagcactta ccaagaacaa tcgaccatgc tcttcaagga acttgtcaaa 3420 ctctcctact gcgtcggggc aatcttagat tatctatacg aaggggcctg ggtccgaata 3480 ccaacacgtc gcagcagctt ttattcactc gcccctaaat gcagcatctc gccatccatc 3540 agaccaagct gtgagaaact actttactct tggattcaat tcctccctct caacgcagtc 3600 taccatccgc cacagttgcc cgccgatgcg gaaggggagt cagccgaaac agtcttcctt 3660 gtccacaaag ccttccttta cttgctgtac ctagcctcca tggcagtcat ataccgcaca 3720 ggcactcata gccaacaaac cagcacggca actccgaaca tggaaggtct acgggcatcg 3780 acgtcacaaa taaaaaaggt ccttgcggag ctgcagggca tgggactgct tcagttcctt 3840 ccaggggcat cagtcactat cctgatgttc gctgtggagg ctagtctgtt ggatctccag 3900 ggctcggata ccatggtccg aaggcaggct atgtcaaatc tatatgcctg tgaggaggct 3960 gcgttacact tgatgcaggc ttatcctacg gctgagatgg cggtattgaa gactaggaca 4020 gcccgtgcaa accttcttgg gttggttgag acatgaagac caatcatgga agctctgctt 4080 ccatcaaagc attgtgtctt tgcttaatga atgaatggat tatgagcgtt tcgcaacctt 4140 ctttaatgca gctacataat agagcataaa cagaggcccg gaaatatata atttggctaa 4200 gaacccggtt gcaagcaggc aagtcacgag gtgcagaaca taccatcaag tttatagatt 4260 attgtagggg atatcctcaa caatatctta cttccaccat tacgtactga agatcgaggt 4320 tatcggggac catgctgtcg tgtcaatagg tacgactcgt t 4361 <210> SEQ ID NO 25 <211> LENGTH: 801 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(801) <400> SEQUENCE: 25 atg cct atc tcc att ccc tcc gcc tcc agc gtc cac gac ctc ttc agc 48 Met Pro Ile Ser Ile Pro Ser Ala Ser Ser Val His Asp Leu Phe Ser 1 5 10 15 ctg aag ggc aag gtc gtc gtt atc acc ggc gct tcc ggc cct cgc ggc 96 Leu Lys Gly Lys Val Val Val Ile Thr Gly Ala Ser Gly Pro Arg Gly 20 25 30 atg ggc att gaa gcc gcg cgt ggc tgc gcc gaa atg ggc gcc aac atc 144 Met Gly Ile Glu Ala Ala Arg Gly Cys Ala Glu Met Gly Ala Asn Ile 35 40 45 gcc ctc acc tac tcc tct cgt cct cag ggt ggt gag aag aac gcc gaa 192 Ala Leu Thr Tyr Ser Ser Arg Pro Gln Gly Gly Glu Lys Asn Ala Glu 50 55 60 gaa ctt cgc aac acc tac ggc gtc aag gcc aag gcc tac cag tgc aac 240 Glu Leu Arg Asn Thr Tyr Gly Val Lys Ala Lys Ala Tyr Gln Cys Asn 65 70 75 80 gtg ggc gac tgg aac agc gtc aag aag ctc gtg gac gac gtg ctg gcc 288 Val Gly Asp Trp Asn Ser Val Lys Lys Leu Val Asp Asp Val Leu Ala 85 90 95 gag ttt ggc cag att gac gcc ttc att gcc aac gct ggc aag aca gcc 336 Glu Phe Gly Gln Ile Asp Ala Phe Ile Ala Asn Ala Gly Lys Thr Ala 100 105 110 agc agt ggt atc ctg gat ggt tcc gtt gag gac tgg gaa gag gtc atc 384 Ser Ser Gly Ile Leu Asp Gly Ser Val Glu Asp Trp Glu Glu Val Ile 115 120 125 cag aca gac ctg acg ggt acc ttc cac tgc gcc aag gcg gtg ggc ccg 432 Gln Thr Asp Leu Thr Gly Thr Phe His Cys Ala Lys Ala Val Gly Pro 130 135 140 cac ttc aag cag cgc gga acg ggc agc ttc atc atc acc tcc agc atg 480 His Phe Lys Gln Arg Gly Thr Gly Ser Phe Ile Ile Thr Ser Ser Met 145 150 155 160 tcg ggt cac att gcc aac ttc ccg cag gag cag acg tcg tac aac gtt 528 Ser Gly His Ile Ala Asn Phe Pro Gln Glu Gln Thr Ser Tyr Asn Val 165 170 175 gcc aag gca ggc tgc atc cat atg gcc cgg tca ttg gcc aac gag tgg 576 Ala Lys Ala Gly Cys Ile His Met Ala Arg Ser Leu Ala Asn Glu Trp 180 185 190 agg gac ttt gcc cgc gtg aac agc atc tcc ccg ggt tac atc gac acg 624 Arg Asp Phe Ala Arg Val Asn Ser Ile Ser Pro Gly Tyr Ile Asp Thr 195 200 205 ggg ctg tcg gac ttt gtg gat aag aag act cag gat ctg tgg atg tcg 672 Gly Leu Ser Asp Phe Val Asp Lys Lys Thr Gln Asp Leu Trp Met Ser 210 215 220 atg atc ccc atg gga cgc aac ggt gat gcc aag gag ctg aag ggt gca 720 Met Ile Pro Met Gly Arg Asn Gly Asp Ala Lys Glu Leu Lys Gly Ala 225 230 235 240 tat gtc tat ctc gcc agt gat gcc agc aca tac acg acg ggt gcc gat 768 Tyr Val Tyr Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp 245 250 255 ttg gtc att gac gga gga tac acc gtg cgg taa 801 Leu Val Ile Asp Gly Gly Tyr Thr Val Arg 260 265 <210> SEQ ID NO 26 <211> LENGTH: 1602 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1602) <400> SEQUENCE: 26 atg agt ggg gta agc ttt gcc cgt cag gcc tta ctc cag gct gct ctt 48 Met Ser Gly Val Ser Phe Ala Arg Gln Ala Leu Leu Gln Ala Ala Leu 1 5 10 15 ctt tcg att gtc tcc gca atc ccg gcg ccc acg gcc ttc gtc aac aat 96 Leu Ser Ile Val Ser Ala Ile Pro Ala Pro Thr Ala Phe Val Asn Asn 20 25 30 gtc gct ccg ccc gag ccc atc atc acc cct tcg ccg gtt caa cat cga 144 Val Ala Pro Pro Glu Pro Ile Ile Thr Pro Ser Pro Val Gln His Arg 35 40 45 cct tct cga gtc gct ggt cga aac att ctg agt gac gtc gat tct gat 192 Pro Ser Arg Val Ala Gly Arg Asn Ile Leu Ser Asp Val Asp Ser Asp 50 55 60 att aac agc atc ctt tct ggc ctt gga tcc gac cta ccc tcc tgg gtc 240 Ile Asn Ser Ile Leu Ser Gly Leu Gly Ser Asp Leu Pro Ser Trp Val 65 70 75 80 gca tcg ggt gtg ccc aac tac ttc cag ggt ttc cct act ggg gat gca 288 Ala Ser Gly Val Pro Asn Tyr Phe Gln Gly Phe Pro Thr Gly Asp Ala 85 90 95 gtg gtg agc tcc ttg ggc ttg aac agc gct gag ttg gca gcc ttg ccc 336 Val Val Ser Ser Leu Gly Leu Asn Ser Ala Glu Leu Ala Ala Leu Pro 100 105 110 acc aat gtc ttg aat atc gac cca tat gcc aac tgg act agc tct ggc 384 Thr Asn Val Leu Asn Ile Asp Pro Tyr Ala Asn Trp Thr Ser Ser Gly 115 120 125 tgg aac gtt cgc ttc cac gga aac gtc tac aag cag ccc aac acc tcc 432 Trp Asn Val Arg Phe His Gly Asn Val Tyr Lys Gln Pro Asn Thr Ser 130 135 140 atc tcc gac ctc aat gat ttg gct gat gtt ttc ctc ggc aat aca agc 480 Ile Ser Asp Leu Asn Asp Leu Ala Asp Val Phe Leu Gly Asn Thr Ser 145 150 155 160 atc tcc gac ctt tcc gaa tcc gag cag aaa cag gct cgg aat tta aca 528 Ile Ser Asp Leu Ser Glu Ser Glu Gln Lys Gln Ala Arg Asn Leu Thr 165 170 175 gcg gaa ata tta gtg gtc cag caa gct cac gtc gcc gta aac aca atc 576 Ala Glu Ile Leu Val Val Gln Gln Ala His Val Ala Val Asn Thr Ile 180 185 190 cac ctg gag cct gca ccg agt cag gga tcc agc gga cag tca ggc ggc 624 His Leu Glu Pro Ala Pro Ser Gln Gly Ser Ser Gly Gln Ser Gly Gly 195 200 205 ggt gga tcc tct aat acc act ggc ggc acc caa gac ctc act ctg ccc 672 Gly Gly Ser Ser Asn Thr Thr Gly Gly Thr Gln Asp Leu Thr Leu Pro 210 215 220 tac aac act aca gtt gaa ggc gat ttt gac act ttc gta ccg att agc 720 Tyr Asn Thr Thr Val Glu Gly Asp Phe Asp Thr Phe Val Pro Ile Ser 225 230 235 240 agc aat ggc ttg acg gca ggt aat gaa act tcg gcg ata caa cgg ctt 768 Ser Asn Gly Leu Thr Ala Gly Asn Glu Thr Ser Ala Ile Gln Arg Leu 245 250 255 aac gtc cat gtg gag ggc gcg aca ata gga aac agt act gcc tat ctc 816 Asn Val His Val Glu Gly Ala Thr Ile Gly Asn Ser Thr Ala Tyr Leu 260 265 270 gta cct ccg acc ggg ctc act gtc gtt tcg gac atc gat gat atc cta 864 Val Pro Pro Thr Gly Leu Thr Val Val Ser Asp Ile Asp Asp Ile Leu 275 280 285 cgc gtc acc aaa atc tat gaa cca gag caa ggt tta ctc aac tca ttt 912 Arg Val Thr Lys Ile Tyr Glu Pro Glu Gln Gly Leu Leu Asn Ser Phe 290 295 300 gcg cgc ccc ttc agg ccg tgg gag aat atg ccg gac atc tat cgc aac 960 Ala Arg Pro Phe Arg Pro Trp Glu Asn Met Pro Asp Ile Tyr Arg Asn 305 310 315 320 tgg tcg atc agt ctc ccc aac ctg cac ttt cac tac cta acg acg acc 1008 Trp Ser Ile Ser Leu Pro Asn Leu His Phe His Tyr Leu Thr Thr Thr 325 330 335

ccc gaa caa gtc acc agg aac tac atg caa ttc atc tac gac aac tac 1056 Pro Glu Gln Val Thr Arg Asn Tyr Met Gln Phe Ile Tyr Asp Asn Tyr 340 345 350 ccc ggt gga tct ttc gac acc cgt ccc ctc aac ttt agc gac gtt tcc 1104 Pro Gly Gly Ser Phe Asp Thr Arg Pro Leu Asn Phe Ser Asp Val Ser 355 360 365 gcc acc ttg tcg atc cgg aaa ttc ctc ctg caa aag gtc ttc gag acc 1152 Ala Thr Leu Ser Ile Arg Lys Phe Leu Leu Gln Lys Val Phe Glu Thr 370 375 380 ttt ccg cag cgc aag ttc atc ctc atc gcc gac acc agc aac agc gac 1200 Phe Pro Gln Arg Lys Phe Ile Leu Ile Ala Asp Thr Ser Asn Ser Asp 385 390 395 400 gtc atg cgt gac tat cct gaa atg gcg acc gac ttc cct ggt caa gtg 1248 Val Met Arg Asp Tyr Pro Glu Met Ala Thr Asp Phe Pro Gly Gln Val 405 410 415 caa tgt att ttt ctc cgg aac acc agc gcc acc gac tca ggg gac aaa 1296 Gln Cys Ile Phe Leu Arg Asn Thr Ser Ala Thr Asp Ser Gly Asp Lys 420 425 430 ttc cca tac gat acc tct ggg ttt aag aag ctc aat cag tca aac tac 1344 Phe Pro Tyr Asp Thr Ser Gly Phe Lys Lys Leu Asn Gln Ser Asn Tyr 435 440 445 atg ttc ttc ttg cat ccc gat gac ttg acg aat cta gac atc gcg aat 1392 Met Phe Phe Leu His Pro Asp Asp Leu Thr Asn Leu Asp Ile Ala Asn 450 455 460 ggc cag tgt tac aac aca tcc att ccg cag aat tta acg ttt agt tat 1440 Gly Gln Cys Tyr Asn Thr Ser Ile Pro Gln Asn Leu Thr Phe Ser Tyr 465 470 475 480 cag ggt ttg cca ttg ggg ctg ggt gat gaa ccc aca gct gtc aat ggg 1488 Gln Gly Leu Pro Leu Gly Leu Gly Asp Glu Pro Thr Ala Val Asn Gly 485 490 495 tct gcc aat cat act gct gag tcg gcg gcc ggt ttg tcg gta aag ggt 1536 Ser Ala Asn His Thr Ala Glu Ser Ala Ala Gly Leu Ser Val Lys Gly 500 505 510 ggg act gat atg cag ggt ttg atc ttt ctg ttc acc ctg gtt acg gcc 1584 Gly Thr Asp Met Gln Gly Leu Ile Phe Leu Phe Thr Leu Val Thr Ala 515 520 525 tat tcc att ttc ttg taa 1602 Tyr Ser Ile Phe Leu 530 <210> SEQ ID NO 27 <211> LENGTH: 1392 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1392) <400> SEQUENCE: 27 atg tcg aag tcg aat ggc gtc agc ctg gca ttc ccc gcc gaa gca gcg 48 Met Ser Lys Ser Asn Gly Val Ser Leu Ala Phe Pro Ala Glu Ala Ala 1 5 10 15 aca aag gaa tat gca gca tct cta gac tct tct gat aga ctt gct gca 96 Thr Lys Glu Tyr Ala Ala Ser Leu Asp Ser Ser Asp Arg Leu Ala Ala 20 25 30 ttt cgg gag aag ttc atc gtc cca tcg aaa gca aat att gct tcc aaa 144 Phe Arg Glu Lys Phe Ile Val Pro Ser Lys Ala Asn Ile Ala Ser Lys 35 40 45 aag cta gca aag cct ggc ctt tcg ccc gag tca tgc att tac ttc tgc 192 Lys Leu Ala Lys Pro Gly Leu Ser Pro Glu Ser Cys Ile Tyr Phe Cys 50 55 60 ggt aac tca ctt ggc att caa ccc aag gcc aca gca aag tac tta gaa 240 Gly Asn Ser Leu Gly Ile Gln Pro Lys Ala Thr Ala Lys Tyr Leu Glu 65 70 75 80 gca cag ttg gac act tgg tca tct att gga gtt ggc ggc cac ttc act 288 Ala Gln Leu Asp Thr Trp Ser Ser Ile Gly Val Gly Gly His Phe Thr 85 90 95 gat ctt gag ggc tca ccc ttg aag caa tgg cag ctg ctc tca gag caa 336 Asp Leu Glu Gly Ser Pro Leu Lys Gln Trp Gln Leu Leu Ser Glu Gln 100 105 110 gca gct gat tca atg agc aag att gtg gga gca aag cca gaa gaa gtc 384 Ala Ala Asp Ser Met Ser Lys Ile Val Gly Ala Lys Pro Glu Glu Val 115 120 125 gca gcg atg gga aca ctt aca acg aac ctt cat ctt ctg ctg gct agt 432 Ala Ala Met Gly Thr Leu Thr Thr Asn Leu His Leu Leu Leu Ala Ser 130 135 140 ttc tat aaa ccg act caa aca aag cac aag atc ttg atg gat tgg aag 480 Phe Tyr Lys Pro Thr Gln Thr Lys His Lys Ile Leu Met Asp Trp Lys 145 150 155 160 gct ttc cca agt gat cac tac gct atc gaa tct cat att gcc tgg cat 528 Ala Phe Pro Ser Asp His Tyr Ala Ile Glu Ser His Ile Ala Trp His 165 170 175 gac ctg gat cct aag gag tct atg gtg ctc atc ggt cca gat gaa ggc 576 Asp Leu Asp Pro Lys Glu Ser Met Val Leu Ile Gly Pro Asp Glu Gly 180 185 190 gaa tac gag ata tcc acc cag aag att ttc tct tat atc gac aag cat 624 Glu Tyr Glu Ile Ser Thr Gln Lys Ile Phe Ser Tyr Ile Asp Lys His 195 200 205 gct gat gag gct gct atg att ctt ctt cct ggt att caa tat tat act 672 Ala Asp Glu Ala Ala Met Ile Leu Leu Pro Gly Ile Gln Tyr Tyr Thr 210 215 220 ggg caa ctg ttc gat atc cag aaa att aca aaa tat gcc cat tcg cgc 720 Gly Gln Leu Phe Asp Ile Gln Lys Ile Thr Lys Tyr Ala His Ser Arg 225 230 235 240 aac atg gtt gtt ggc tgg gat ctg gcc cat gcg ttc gct aat gtt gag 768 Asn Met Val Val Gly Trp Asp Leu Ala His Ala Phe Ala Asn Val Glu 245 250 255 cta aag ctg cat gat tgg aac gtt gat ttt gca gca tgg tgc aca tat 816 Leu Lys Leu His Asp Trp Asn Val Asp Phe Ala Ala Trp Cys Thr Tyr 260 265 270 aag tac gga aac gcc ggt cct gga gca atg ggg ggg ctt ttc gtg cat 864 Lys Tyr Gly Asn Ala Gly Pro Gly Ala Met Gly Gly Leu Phe Val His 275 280 285 gaa caa cac gga gag gtc gat tac agc gcg gga gaa gat gca ccc aaa 912 Glu Gln His Gly Glu Val Asp Tyr Ser Ala Gly Glu Asp Ala Pro Lys 290 295 300 ttc cgc cat cgt ctg acc ggg tgg tat ggt ggc gat cga tcg gta aga 960 Phe Arg His Arg Leu Thr Gly Trp Tyr Gly Gly Asp Arg Ser Val Arg 305 310 315 320 ttc aag atg gac aac aag ttc aaa cct att cct ggg gca gga gga ttt 1008 Phe Lys Met Asp Asn Lys Phe Lys Pro Ile Pro Gly Ala Gly Gly Phe 325 330 335 cag ata tca aat cct tcg gcc atc gac ctt gcg tgt ctt tgt gcc gct 1056 Gln Ile Ser Asn Pro Ser Ala Ile Asp Leu Ala Cys Leu Cys Ala Ala 340 345 350 tta tcg gtg ttt gat gag acg tca atg gca gat ctt cgc aga aaa tca 1104 Leu Ser Val Phe Asp Glu Thr Ser Met Ala Asp Leu Arg Arg Lys Ser 355 360 365 ttg aag cta aca gca tac ctt gag ttc ctt ttg ctc agg gat tat gaa 1152 Leu Lys Leu Thr Ala Tyr Leu Glu Phe Leu Leu Leu Arg Asp Tyr Glu 370 375 380 gaa gaa tct agg cca ttc agc att atc acg ccc aag gac ccc gag gcg 1200 Glu Glu Ser Arg Pro Phe Ser Ile Ile Thr Pro Lys Asp Pro Glu Ala 385 390 395 400 aga ggc gca caa ctg agt cta ttg ctc aag cct ggt cta tta cag aat 1248 Arg Gly Ala Gln Leu Ser Leu Leu Leu Lys Pro Gly Leu Leu Gln Asn 405 410 415 gtt gca cag aag ttg caa gaa gca gga ata gtc tgc gat aaa cgg gag 1296 Val Ala Gln Lys Leu Gln Glu Ala Gly Ile Val Cys Asp Lys Arg Glu 420 425 430 ccg ggt gtt gtg cgt gtc gct ccg gtt cct ttg tac aac agc ttc agc 1344 Pro Gly Val Val Arg Val Ala Pro Val Pro Leu Tyr Asn Ser Phe Ser 435 440 445 gag gtt tgg aca ttt gtg aag atc ttc aag gat gca ctg cag cag tga 1392 Glu Val Trp Thr Phe Val Lys Ile Phe Lys Asp Ala Leu Gln Gln 450 455 460 <210> SEQ ID NO 28 <211> LENGTH: 1143 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1143) <400> SEQUENCE: 28 atg gat gtg act aat ttg gct gtc gat tgt ccc gtt ata gag acc ggt 48 Met Asp Val Thr Asn Leu Ala Val Asp Cys Pro Val Ile Glu Thr Gly 1 5 10 15 tcc ctt gct ttg tgg atg gct tcc tcc gaa gtg gac cag aag gcc ttg 96 Ser Leu Ala Leu Trp Met Ala Ser Ser Glu Val Asp Gln Lys Ala Leu 20 25 30 ggc aac ttt gcg gcc ata acg ggc ctc gag aac ccg ttc ctg tcc gcg 144 Gly Asn Phe Ala Ala Ile Thr Gly Leu Glu Asn Pro Phe Leu Ser Ala 35 40 45 atg ctg tac aag att ctg ggg ttg tct gtt atg tta tcg aag aaa ctc 192 Met Leu Tyr Lys Ile Leu Gly Leu Ser Val Met Leu Ser Lys Lys Leu 50 55 60 ctc cgt gca cga cga ttg cgg cgc cta gac ccc acc cga gag act aaa 240 Leu Arg Ala Arg Arg Leu Arg Arg Leu Asp Pro Thr Arg Glu Thr Lys 65 70 75 80 tcg ctt cat ctt tat tac cat atc atc tgg ctt tcg cgt gag ggt ctt 288 Ser Leu His Leu Tyr Tyr His Ile Ile Trp Leu Ser Arg Glu Gly Leu 85 90 95 ctg ata tta gag gag ttc gtc ctg cca ctg gtt gag gga ttt atg gaa 336 Leu Ile Leu Glu Glu Phe Val Leu Pro Leu Val Glu Gly Phe Met Glu 100 105 110 ctc aaa att tta gct tac aag ctg cgc gcc tcc ttt tac cat ata ttc 384 Leu Lys Ile Leu Ala Tyr Lys Leu Arg Ala Ser Phe Tyr His Ile Phe 115 120 125 gtc cta ttt cag aac cag cct gct gtc cac tcg ccg ggt atc gtt agt 432 Val Leu Phe Gln Asn Gln Pro Ala Val His Ser Pro Gly Ile Val Ser 130 135 140 ctg ccg agt agt aca cct tta tcc aac ggc gtg aca gaa gcg gag tca 480 Leu Pro Ser Ser Thr Pro Leu Ser Asn Gly Val Thr Glu Ala Glu Ser 145 150 155 160 cca tcc aag gat tcc aat ttg aga ttt tca ttt cag ctg aag ccc gac 528 Pro Ser Lys Asp Ser Asn Leu Arg Phe Ser Phe Gln Leu Lys Pro Asp 165 170 175 acg ata acg gtt tcc ggg aag ccc agt tcc gcc tcg gat agt gcg cca 576 Thr Ile Thr Val Ser Gly Lys Pro Ser Ser Ala Ser Asp Ser Ala Pro 180 185 190 cga ggc aag gtt acg cag gca ccc ccg ggc ttt gcg ccg gtt caa ccg 624 Arg Gly Lys Val Thr Gln Ala Pro Pro Gly Phe Ala Pro Val Gln Pro 195 200 205 ccc aag tcg aac tcg gcg ttt ctc ctc cct gct ctc gat tat acc cct 672 Pro Lys Ser Asn Ser Ala Phe Leu Leu Pro Ala Leu Asp Tyr Thr Pro 210 215 220 aca gct acc gca tgc ttc aat cat gcc gcc ctc ttg gca gat caa ttc 720 Thr Ala Thr Ala Cys Phe Asn His Ala Ala Leu Leu Ala Asp Gln Phe 225 230 235 240

ctc ccg ggg tcg cac ccg ctc cgt ctg tcc atc aag ctt gaa ttt gcc 768 Leu Pro Gly Ser His Pro Leu Arg Leu Ser Ile Lys Leu Glu Phe Ala 245 250 255 gcc tac ctc tac gat tgc cta cat gac gct cat gcc tgt cga cgg ttg 816 Ala Tyr Leu Tyr Asp Cys Leu His Asp Ala His Ala Cys Arg Arg Leu 260 265 270 gcc aag caa gcc att gcc gat gtg tac aat gcg caa gag ggc atg gac 864 Ala Lys Gln Ala Ile Ala Asp Val Tyr Asn Ala Gln Glu Gly Met Asp 275 280 285 gac gag agc ttc gag gat gca gct gag att gtg ggt att ttg ggc aag 912 Asp Glu Ser Phe Glu Asp Ala Ala Glu Ile Val Gly Ile Leu Gly Lys 290 295 300 atg gtg aag cgg gga agg aac aca agc agt gct gga aat agc acc acg 960 Met Val Lys Arg Gly Arg Asn Thr Ser Ser Ala Gly Asn Ser Thr Thr 305 310 315 320 gct gtg aaa acg ccc cag gag gag cga agt gag ggc agt cga aca cca 1008 Ala Val Lys Thr Pro Gln Glu Glu Arg Ser Glu Gly Ser Arg Thr Pro 325 330 335 tcc tct cag aca acg ttg aaa agg ccg gcg cga gtg ccg aaa agc acc 1056 Ser Ser Gln Thr Thr Leu Lys Arg Pro Ala Arg Val Pro Lys Ser Thr 340 345 350 tcc tca ccg gcg cgc gcg acc aag ccg acg act agc gag ggg atg tcc 1104 Ser Ser Pro Ala Arg Ala Thr Lys Pro Thr Thr Ser Glu Gly Met Ser 355 360 365 ccc gct gtg ccc gac ccc acc atg atg aat ccc ata tga 1143 Pro Ala Val Pro Asp Pro Thr Met Met Asn Pro Ile 370 375 380 <210> SEQ ID NO 29 <211> LENGTH: 1779 <212> TYPE: DNA <213> ORGANISM: Aspergillus niger <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1779) <400> SEQUENCE: 29 atg aga aat ttg gag tgt gtc tac aca aaa tca cga agg tat ccc tcg 48 Met Arg Asn Leu Glu Cys Val Tyr Thr Lys Ser Arg Arg Tyr Pro Ser 1 5 10 15 tct atc ttt ccg aca ctc agg agg tcg tca act agt ccg aac acg gcg 96 Ser Ile Phe Pro Thr Leu Arg Arg Ser Ser Thr Ser Pro Asn Thr Ala 20 25 30 tcc tct cct gac act aat gcc tca tct tcg cca tgc tcg gac tct agt 144 Ser Ser Pro Asp Thr Asn Ala Ser Ser Ser Pro Cys Ser Asp Ser Ser 35 40 45 ggg acc cat gct ggc att tcc att ccc cac tca gat agt ccc gcc agt 192 Gly Thr His Ala Gly Ile Ser Ile Pro His Ser Asp Ser Pro Ala Ser 50 55 60 gtg gac gaa tgt ggg aac aac ggc ctt ttc gac ggg gaa tct cta act 240 Val Asp Glu Cys Gly Asn Asn Gly Leu Phe Asp Gly Glu Ser Leu Thr 65 70 75 80 caa tta ttg gtc tct ggg agc aaa gcc ata tca ggc caa cca cat atc 288 Gln Leu Leu Val Ser Gly Ser Lys Ala Ile Ser Gly Gln Pro His Ile 85 90 95 gcc att aag cca cat gtt ctg tct acc tat ccc ctt gac aac ctc ctc 336 Ala Ile Lys Pro His Val Leu Ser Thr Tyr Pro Leu Asp Asn Leu Leu 100 105 110 tca gac att gag gga ctc ctc gag cag cca gtc tct cat ttg gct caa 384 Ser Asp Ile Glu Gly Leu Leu Glu Gln Pro Val Ser His Leu Ala Gln 115 120 125 agc aag gaa gat cta aca aaa ccg gac cgg atg ttt cga act cgc ctt 432 Ser Lys Glu Asp Leu Thr Lys Pro Asp Arg Met Phe Arg Thr Arg Leu 130 135 140 cca gcg gtc tca ggt gag gac aat gag tat ctg cta agg aaa gga gct 480 Pro Ala Val Ser Gly Glu Asp Asn Glu Tyr Leu Leu Arg Lys Gly Ala 145 150 155 160 ctg ctg ctc ttg cca cca gcc ttg cgc aac gaa cta ttg gcg gca tat 528 Leu Leu Leu Leu Pro Pro Ala Leu Arg Asn Glu Leu Leu Ala Ala Tyr 165 170 175 gtc aac tac gtc cat ccg cta tta ccg att ctt gat ctt gga gac ttt 576 Val Asn Tyr Val His Pro Leu Leu Pro Ile Leu Asp Leu Gly Asp Phe 180 185 190 ctc tca caa atc att gtt gga gat gat gct caa ttc aag tca cca cta 624 Leu Ser Gln Ile Ile Val Gly Asp Asp Ala Gln Phe Lys Ser Pro Leu 195 200 205 tta tac caa gct gtc atg ttc gcg ggc agt att ttt gct cag cga gac 672 Leu Tyr Gln Ala Val Met Phe Ala Gly Ser Ile Phe Ala Gln Arg Asp 210 215 220 ggc aca gac gaa cct gaa cag cta acg agg gct tta ttt gaa cgt aca 720 Gly Thr Asp Glu Pro Glu Gln Leu Thr Arg Ala Leu Phe Glu Arg Thr 225 230 235 240 aag gtt cta tac gag ttt gaa tcc gaa tca tgt gcc tac act caa att 768 Lys Val Leu Tyr Glu Phe Glu Ser Glu Ser Cys Ala Tyr Thr Gln Ile 245 250 255 caa gct ctc tta ttg atg act ctc tgg cat gga gat atg tca cgc cac 816 Gln Ala Leu Leu Leu Met Thr Leu Trp His Gly Asp Met Ser Arg His 260 265 270 aaa ggc ccg tct tac tgg ctt gac gtc gcc ttc tcc act gcg gaa aga 864 Lys Gly Pro Ser Tyr Trp Leu Asp Val Ala Phe Ser Thr Ala Glu Arg 275 280 285 att ggc cta ctc cat gat gaa gac tgg cca tgc gat agc cgt tcc ttc 912 Ile Gly Leu Leu His Asp Glu Asp Trp Pro Cys Asp Ser Arg Ser Phe 290 295 300 cga tcc gct atg tgg tgt tgc ctt tac gta cgt gat cga aca atc tcg 960 Arg Ser Ala Met Trp Cys Cys Leu Tyr Val Arg Asp Arg Thr Ile Ser 305 310 315 320 ctt ggg tct cgt cgg cct cca cgg atg cca gtc aac aaa tat ccg gac 1008 Leu Gly Ser Arg Arg Pro Pro Arg Met Pro Val Asn Lys Tyr Pro Asp 325 330 335 tca att ctc cat ctt atg agc gac gca cct cgg gaa tat gac gaa ata 1056 Ser Ile Leu His Leu Met Ser Asp Ala Pro Arg Glu Tyr Asp Glu Ile 340 345 350 att ctt gag agt cta ggt agc gct tct ctg atg ctt aca agc act tac 1104 Ile Leu Glu Ser Leu Gly Ser Ala Ser Leu Met Leu Thr Ser Thr Tyr 355 360 365 caa gaa caa tcg acc atg ctc ttc aag gaa ctt gtc aaa ctc tcc tac 1152 Gln Glu Gln Ser Thr Met Leu Phe Lys Glu Leu Val Lys Leu Ser Tyr 370 375 380 tgc gtc ggg gca atc tta gat tat cta tac gaa ggg gcc tgg gtc cga 1200 Cys Val Gly Ala Ile Leu Asp Tyr Leu Tyr Glu Gly Ala Trp Val Arg 385 390 395 400 ata cca aca cgt cgc agc agc ttt tat tca ctc gcc cct aaa tgc agc 1248 Ile Pro Thr Arg Arg Ser Ser Phe Tyr Ser Leu Ala Pro Lys Cys Ser 405 410 415 atc tcg cca tcc atc aga cca agc tgt gag aaa cta ctt tac tct tgg 1296 Ile Ser Pro Ser Ile Arg Pro Ser Cys Glu Lys Leu Leu Tyr Ser Trp 420 425 430 att caa ttc ctc cct ctc aac gca gtc tac cat ccg cca cag ttg ccc 1344 Ile Gln Phe Leu Pro Leu Asn Ala Val Tyr His Pro Pro Gln Leu Pro 435 440 445 gcc gat gcg gaa ggg gag tca gcc gaa aca gtc ttc ctt gtc cac aaa 1392 Ala Asp Ala Glu Gly Glu Ser Ala Glu Thr Val Phe Leu Val His Lys 450 455 460 gcc ttc ctt tac ttg ctg tac cta gcc tcc atg gca gtc ata tac cgc 1440 Ala Phe Leu Tyr Leu Leu Tyr Leu Ala Ser Met Ala Val Ile Tyr Arg 465 470 475 480 aca ggc act cat agc caa caa acc agc acg gca act ccg aac atg gaa 1488 Thr Gly Thr His Ser Gln Gln Thr Ser Thr Ala Thr Pro Asn Met Glu 485 490 495 ggt cta cgg gca tcg acg tca caa ata aaa aag gtc ctt gcg gag ctg 1536 Gly Leu Arg Ala Ser Thr Ser Gln Ile Lys Lys Val Leu Ala Glu Leu 500 505 510 cag ggc atg gga ctg ctt cag ttc ctt cca ggg gca tca gtc act atc 1584 Gln Gly Met Gly Leu Leu Gln Phe Leu Pro Gly Ala Ser Val Thr Ile 515 520 525 ctg atg ttc gct gtg gag gct agt ctg ttg gat ctc cag ggc tcg gat 1632 Leu Met Phe Ala Val Glu Ala Ser Leu Leu Asp Leu Gln Gly Ser Asp 530 535 540 acc atg gtc cga agg cag gct atg tca aat cta tat gcc tgt gag gag 1680 Thr Met Val Arg Arg Gln Ala Met Ser Asn Leu Tyr Ala Cys Glu Glu 545 550 555 560 gct gcg tta cac ttg atg cag gct tat cct acg gct gag atg gcg gta 1728 Ala Ala Leu His Leu Met Gln Ala Tyr Pro Thr Ala Glu Met Ala Val 565 570 575 ttg aag act agg aca gcc cgt gca aac ctt ctt ggg ttg gtt gag aca 1776 Leu Lys Thr Arg Thr Ala Arg Ala Asn Leu Leu Gly Leu Val Glu Thr 580 585 590 tga 1779 <210> SEQ ID NO 30 <211> LENGTH: 266 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 30 Met Pro Ile Ser Ile Pro Ser Ala Ser Ser Val His Asp Leu Phe Ser 1 5 10 15 Leu Lys Gly Lys Val Val Val Ile Thr Gly Ala Ser Gly Pro Arg Gly 20 25 30 Met Gly Ile Glu Ala Ala Arg Gly Cys Ala Glu Met Gly Ala Asn Ile 35 40 45 Ala Leu Thr Tyr Ser Ser Arg Pro Gln Gly Gly Glu Lys Asn Ala Glu 50 55 60 Glu Leu Arg Asn Thr Tyr Gly Val Lys Ala Lys Ala Tyr Gln Cys Asn 65 70 75 80 Val Gly Asp Trp Asn Ser Val Lys Lys Leu Val Asp Asp Val Leu Ala 85 90 95 Glu Phe Gly Gln Ile Asp Ala Phe Ile Ala Asn Ala Gly Lys Thr Ala 100 105 110 Ser Ser Gly Ile Leu Asp Gly Ser Val Glu Asp Trp Glu Glu Val Ile 115 120 125 Gln Thr Asp Leu Thr Gly Thr Phe His Cys Ala Lys Ala Val Gly Pro 130 135 140 His Phe Lys Gln Arg Gly Thr Gly Ser Phe Ile Ile Thr Ser Ser Met 145 150 155 160 Ser Gly His Ile Ala Asn Phe Pro Gln Glu Gln Thr Ser Tyr Asn Val 165 170 175 Ala Lys Ala Gly Cys Ile His Met Ala Arg Ser Leu Ala Asn Glu Trp 180 185 190 Arg Asp Phe Ala Arg Val Asn Ser Ile Ser Pro Gly Tyr Ile Asp Thr 195 200 205 Gly Leu Ser Asp Phe Val Asp Lys Lys Thr Gln Asp Leu Trp Met Ser 210 215 220 Met Ile Pro Met Gly Arg Asn Gly Asp Ala Lys Glu Leu Lys Gly Ala 225 230 235 240 Tyr Val Tyr Leu Ala Ser Asp Ala Ser Thr Tyr Thr Thr Gly Ala Asp

245 250 255 Leu Val Ile Asp Gly Gly Tyr Thr Val Arg 260 265 <210> SEQ ID NO 31 <211> LENGTH: 533 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 31 Met Ser Gly Val Ser Phe Ala Arg Gln Ala Leu Leu Gln Ala Ala Leu 1 5 10 15 Leu Ser Ile Val Ser Ala Ile Pro Ala Pro Thr Ala Phe Val Asn Asn 20 25 30 Val Ala Pro Pro Glu Pro Ile Ile Thr Pro Ser Pro Val Gln His Arg 35 40 45 Pro Ser Arg Val Ala Gly Arg Asn Ile Leu Ser Asp Val Asp Ser Asp 50 55 60 Ile Asn Ser Ile Leu Ser Gly Leu Gly Ser Asp Leu Pro Ser Trp Val 65 70 75 80 Ala Ser Gly Val Pro Asn Tyr Phe Gln Gly Phe Pro Thr Gly Asp Ala 85 90 95 Val Val Ser Ser Leu Gly Leu Asn Ser Ala Glu Leu Ala Ala Leu Pro 100 105 110 Thr Asn Val Leu Asn Ile Asp Pro Tyr Ala Asn Trp Thr Ser Ser Gly 115 120 125 Trp Asn Val Arg Phe His Gly Asn Val Tyr Lys Gln Pro Asn Thr Ser 130 135 140 Ile Ser Asp Leu Asn Asp Leu Ala Asp Val Phe Leu Gly Asn Thr Ser 145 150 155 160 Ile Ser Asp Leu Ser Glu Ser Glu Gln Lys Gln Ala Arg Asn Leu Thr 165 170 175 Ala Glu Ile Leu Val Val Gln Gln Ala His Val Ala Val Asn Thr Ile 180 185 190 His Leu Glu Pro Ala Pro Ser Gln Gly Ser Ser Gly Gln Ser Gly Gly 195 200 205 Gly Gly Ser Ser Asn Thr Thr Gly Gly Thr Gln Asp Leu Thr Leu Pro 210 215 220 Tyr Asn Thr Thr Val Glu Gly Asp Phe Asp Thr Phe Val Pro Ile Ser 225 230 235 240 Ser Asn Gly Leu Thr Ala Gly Asn Glu Thr Ser Ala Ile Gln Arg Leu 245 250 255 Asn Val His Val Glu Gly Ala Thr Ile Gly Asn Ser Thr Ala Tyr Leu 260 265 270 Val Pro Pro Thr Gly Leu Thr Val Val Ser Asp Ile Asp Asp Ile Leu 275 280 285 Arg Val Thr Lys Ile Tyr Glu Pro Glu Gln Gly Leu Leu Asn Ser Phe 290 295 300 Ala Arg Pro Phe Arg Pro Trp Glu Asn Met Pro Asp Ile Tyr Arg Asn 305 310 315 320 Trp Ser Ile Ser Leu Pro Asn Leu His Phe His Tyr Leu Thr Thr Thr 325 330 335 Pro Glu Gln Val Thr Arg Asn Tyr Met Gln Phe Ile Tyr Asp Asn Tyr 340 345 350 Pro Gly Gly Ser Phe Asp Thr Arg Pro Leu Asn Phe Ser Asp Val Ser 355 360 365 Ala Thr Leu Ser Ile Arg Lys Phe Leu Leu Gln Lys Val Phe Glu Thr 370 375 380 Phe Pro Gln Arg Lys Phe Ile Leu Ile Ala Asp Thr Ser Asn Ser Asp 385 390 395 400 Val Met Arg Asp Tyr Pro Glu Met Ala Thr Asp Phe Pro Gly Gln Val 405 410 415 Gln Cys Ile Phe Leu Arg Asn Thr Ser Ala Thr Asp Ser Gly Asp Lys 420 425 430 Phe Pro Tyr Asp Thr Ser Gly Phe Lys Lys Leu Asn Gln Ser Asn Tyr 435 440 445 Met Phe Phe Leu His Pro Asp Asp Leu Thr Asn Leu Asp Ile Ala Asn 450 455 460 Gly Gln Cys Tyr Asn Thr Ser Ile Pro Gln Asn Leu Thr Phe Ser Tyr 465 470 475 480 Gln Gly Leu Pro Leu Gly Leu Gly Asp Glu Pro Thr Ala Val Asn Gly 485 490 495 Ser Ala Asn His Thr Ala Glu Ser Ala Ala Gly Leu Ser Val Lys Gly 500 505 510 Gly Thr Asp Met Gln Gly Leu Ile Phe Leu Phe Thr Leu Val Thr Ala 515 520 525 Tyr Ser Ile Phe Leu 530 <210> SEQ ID NO 32 <211> LENGTH: 463 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 32 Met Ser Lys Ser Asn Gly Val Ser Leu Ala Phe Pro Ala Glu Ala Ala 1 5 10 15 Thr Lys Glu Tyr Ala Ala Ser Leu Asp Ser Ser Asp Arg Leu Ala Ala 20 25 30 Phe Arg Glu Lys Phe Ile Val Pro Ser Lys Ala Asn Ile Ala Ser Lys 35 40 45 Lys Leu Ala Lys Pro Gly Leu Ser Pro Glu Ser Cys Ile Tyr Phe Cys 50 55 60 Gly Asn Ser Leu Gly Ile Gln Pro Lys Ala Thr Ala Lys Tyr Leu Glu 65 70 75 80 Ala Gln Leu Asp Thr Trp Ser Ser Ile Gly Val Gly Gly His Phe Thr 85 90 95 Asp Leu Glu Gly Ser Pro Leu Lys Gln Trp Gln Leu Leu Ser Glu Gln 100 105 110 Ala Ala Asp Ser Met Ser Lys Ile Val Gly Ala Lys Pro Glu Glu Val 115 120 125 Ala Ala Met Gly Thr Leu Thr Thr Asn Leu His Leu Leu Leu Ala Ser 130 135 140 Phe Tyr Lys Pro Thr Gln Thr Lys His Lys Ile Leu Met Asp Trp Lys 145 150 155 160 Ala Phe Pro Ser Asp His Tyr Ala Ile Glu Ser His Ile Ala Trp His 165 170 175 Asp Leu Asp Pro Lys Glu Ser Met Val Leu Ile Gly Pro Asp Glu Gly 180 185 190 Glu Tyr Glu Ile Ser Thr Gln Lys Ile Phe Ser Tyr Ile Asp Lys His 195 200 205 Ala Asp Glu Ala Ala Met Ile Leu Leu Pro Gly Ile Gln Tyr Tyr Thr 210 215 220 Gly Gln Leu Phe Asp Ile Gln Lys Ile Thr Lys Tyr Ala His Ser Arg 225 230 235 240 Asn Met Val Val Gly Trp Asp Leu Ala His Ala Phe Ala Asn Val Glu 245 250 255 Leu Lys Leu His Asp Trp Asn Val Asp Phe Ala Ala Trp Cys Thr Tyr 260 265 270 Lys Tyr Gly Asn Ala Gly Pro Gly Ala Met Gly Gly Leu Phe Val His 275 280 285 Glu Gln His Gly Glu Val Asp Tyr Ser Ala Gly Glu Asp Ala Pro Lys 290 295 300 Phe Arg His Arg Leu Thr Gly Trp Tyr Gly Gly Asp Arg Ser Val Arg 305 310 315 320 Phe Lys Met Asp Asn Lys Phe Lys Pro Ile Pro Gly Ala Gly Gly Phe 325 330 335 Gln Ile Ser Asn Pro Ser Ala Ile Asp Leu Ala Cys Leu Cys Ala Ala 340 345 350 Leu Ser Val Phe Asp Glu Thr Ser Met Ala Asp Leu Arg Arg Lys Ser 355 360 365 Leu Lys Leu Thr Ala Tyr Leu Glu Phe Leu Leu Leu Arg Asp Tyr Glu 370 375 380 Glu Glu Ser Arg Pro Phe Ser Ile Ile Thr Pro Lys Asp Pro Glu Ala 385 390 395 400 Arg Gly Ala Gln Leu Ser Leu Leu Leu Lys Pro Gly Leu Leu Gln Asn 405 410 415 Val Ala Gln Lys Leu Gln Glu Ala Gly Ile Val Cys Asp Lys Arg Glu 420 425 430 Pro Gly Val Val Arg Val Ala Pro Val Pro Leu Tyr Asn Ser Phe Ser 435 440 445 Glu Val Trp Thr Phe Val Lys Ile Phe Lys Asp Ala Leu Gln Gln 450 455 460 <210> SEQ ID NO 33 <211> LENGTH: 380 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 33 Met Asp Val Thr Asn Leu Ala Val Asp Cys Pro Val Ile Glu Thr Gly 1 5 10 15 Ser Leu Ala Leu Trp Met Ala Ser Ser Glu Val Asp Gln Lys Ala Leu 20 25 30 Gly Asn Phe Ala Ala Ile Thr Gly Leu Glu Asn Pro Phe Leu Ser Ala 35 40 45 Met Leu Tyr Lys Ile Leu Gly Leu Ser Val Met Leu Ser Lys Lys Leu 50 55 60 Leu Arg Ala Arg Arg Leu Arg Arg Leu Asp Pro Thr Arg Glu Thr Lys 65 70 75 80 Ser Leu His Leu Tyr Tyr His Ile Ile Trp Leu Ser Arg Glu Gly Leu 85 90 95 Leu Ile Leu Glu Glu Phe Val Leu Pro Leu Val Glu Gly Phe Met Glu 100 105 110 Leu Lys Ile Leu Ala Tyr Lys Leu Arg Ala Ser Phe Tyr His Ile Phe 115 120 125 Val Leu Phe Gln Asn Gln Pro Ala Val His Ser Pro Gly Ile Val Ser 130 135 140 Leu Pro Ser Ser Thr Pro Leu Ser Asn Gly Val Thr Glu Ala Glu Ser 145 150 155 160 Pro Ser Lys Asp Ser Asn Leu Arg Phe Ser Phe Gln Leu Lys Pro Asp 165 170 175

Thr Ile Thr Val Ser Gly Lys Pro Ser Ser Ala Ser Asp Ser Ala Pro 180 185 190 Arg Gly Lys Val Thr Gln Ala Pro Pro Gly Phe Ala Pro Val Gln Pro 195 200 205 Pro Lys Ser Asn Ser Ala Phe Leu Leu Pro Ala Leu Asp Tyr Thr Pro 210 215 220 Thr Ala Thr Ala Cys Phe Asn His Ala Ala Leu Leu Ala Asp Gln Phe 225 230 235 240 Leu Pro Gly Ser His Pro Leu Arg Leu Ser Ile Lys Leu Glu Phe Ala 245 250 255 Ala Tyr Leu Tyr Asp Cys Leu His Asp Ala His Ala Cys Arg Arg Leu 260 265 270 Ala Lys Gln Ala Ile Ala Asp Val Tyr Asn Ala Gln Glu Gly Met Asp 275 280 285 Asp Glu Ser Phe Glu Asp Ala Ala Glu Ile Val Gly Ile Leu Gly Lys 290 295 300 Met Val Lys Arg Gly Arg Asn Thr Ser Ser Ala Gly Asn Ser Thr Thr 305 310 315 320 Ala Val Lys Thr Pro Gln Glu Glu Arg Ser Glu Gly Ser Arg Thr Pro 325 330 335 Ser Ser Gln Thr Thr Leu Lys Arg Pro Ala Arg Val Pro Lys Ser Thr 340 345 350 Ser Ser Pro Ala Arg Ala Thr Lys Pro Thr Thr Ser Glu Gly Met Ser 355 360 365 Pro Ala Val Pro Asp Pro Thr Met Met Asn Pro Ile 370 375 380 <210> SEQ ID NO 34 <211> LENGTH: 592 <212> TYPE: PRT <213> ORGANISM: Aspergillus niger <400> SEQUENCE: 34 Met Arg Asn Leu Glu Cys Val Tyr Thr Lys Ser Arg Arg Tyr Pro Ser 1 5 10 15 Ser Ile Phe Pro Thr Leu Arg Arg Ser Ser Thr Ser Pro Asn Thr Ala 20 25 30 Ser Ser Pro Asp Thr Asn Ala Ser Ser Ser Pro Cys Ser Asp Ser Ser 35 40 45 Gly Thr His Ala Gly Ile Ser Ile Pro His Ser Asp Ser Pro Ala Ser 50 55 60 Val Asp Glu Cys Gly Asn Asn Gly Leu Phe Asp Gly Glu Ser Leu Thr 65 70 75 80 Gln Leu Leu Val Ser Gly Ser Lys Ala Ile Ser Gly Gln Pro His Ile 85 90 95 Ala Ile Lys Pro His Val Leu Ser Thr Tyr Pro Leu Asp Asn Leu Leu 100 105 110 Ser Asp Ile Glu Gly Leu Leu Glu Gln Pro Val Ser His Leu Ala Gln 115 120 125 Ser Lys Glu Asp Leu Thr Lys Pro Asp Arg Met Phe Arg Thr Arg Leu 130 135 140 Pro Ala Val Ser Gly Glu Asp Asn Glu Tyr Leu Leu Arg Lys Gly Ala 145 150 155 160 Leu Leu Leu Leu Pro Pro Ala Leu Arg Asn Glu Leu Leu Ala Ala Tyr 165 170 175 Val Asn Tyr Val His Pro Leu Leu Pro Ile Leu Asp Leu Gly Asp Phe 180 185 190 Leu Ser Gln Ile Ile Val Gly Asp Asp Ala Gln Phe Lys Ser Pro Leu 195 200 205 Leu Tyr Gln Ala Val Met Phe Ala Gly Ser Ile Phe Ala Gln Arg Asp 210 215 220 Gly Thr Asp Glu Pro Glu Gln Leu Thr Arg Ala Leu Phe Glu Arg Thr 225 230 235 240 Lys Val Leu Tyr Glu Phe Glu Ser Glu Ser Cys Ala Tyr Thr Gln Ile 245 250 255 Gln Ala Leu Leu Leu Met Thr Leu Trp His Gly Asp Met Ser Arg His 260 265 270 Lys Gly Pro Ser Tyr Trp Leu Asp Val Ala Phe Ser Thr Ala Glu Arg 275 280 285 Ile Gly Leu Leu His Asp Glu Asp Trp Pro Cys Asp Ser Arg Ser Phe 290 295 300 Arg Ser Ala Met Trp Cys Cys Leu Tyr Val Arg Asp Arg Thr Ile Ser 305 310 315 320 Leu Gly Ser Arg Arg Pro Pro Arg Met Pro Val Asn Lys Tyr Pro Asp 325 330 335 Ser Ile Leu His Leu Met Ser Asp Ala Pro Arg Glu Tyr Asp Glu Ile 340 345 350 Ile Leu Glu Ser Leu Gly Ser Ala Ser Leu Met Leu Thr Ser Thr Tyr 355 360 365 Gln Glu Gln Ser Thr Met Leu Phe Lys Glu Leu Val Lys Leu Ser Tyr 370 375 380 Cys Val Gly Ala Ile Leu Asp Tyr Leu Tyr Glu Gly Ala Trp Val Arg 385 390 395 400 Ile Pro Thr Arg Arg Ser Ser Phe Tyr Ser Leu Ala Pro Lys Cys Ser 405 410 415 Ile Ser Pro Ser Ile Arg Pro Ser Cys Glu Lys Leu Leu Tyr Ser Trp 420 425 430 Ile Gln Phe Leu Pro Leu Asn Ala Val Tyr His Pro Pro Gln Leu Pro 435 440 445 Ala Asp Ala Glu Gly Glu Ser Ala Glu Thr Val Phe Leu Val His Lys 450 455 460 Ala Phe Leu Tyr Leu Leu Tyr Leu Ala Ser Met Ala Val Ile Tyr Arg 465 470 475 480 Thr Gly Thr His Ser Gln Gln Thr Ser Thr Ala Thr Pro Asn Met Glu 485 490 495 Gly Leu Arg Ala Ser Thr Ser Gln Ile Lys Lys Val Leu Ala Glu Leu 500 505 510 Gln Gly Met Gly Leu Leu Gln Phe Leu Pro Gly Ala Ser Val Thr Ile 515 520 525 Leu Met Phe Ala Val Glu Ala Ser Leu Leu Asp Leu Gln Gly Ser Asp 530 535 540 Thr Met Val Arg Arg Gln Ala Met Ser Asn Leu Tyr Ala Cys Glu Glu 545 550 555 560 Ala Ala Leu His Leu Met Gln Ala Tyr Pro Thr Ala Glu Met Ala Val 565 570 575 Leu Lys Thr Arg Thr Ala Arg Ala Asn Leu Leu Gly Leu Val Glu Thr 580 585 590 <210> SEQ ID NO 35 <211> LENGTH: 1076 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(35) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1062) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1037)..(1076) <400> SEQUENCE: 35 acttcactcg agataatgta ggcgacaaag tagccgggca tgcgaccgga ggactcggtc 60 agaggcacga atatgagagg ccagaacgcc gcacccatgt tccaagatgt tgtggcccag 120 taaaggttag gaaatggggt gttttgaacg tggaatcgtt cttccatcca gttgttgccg 180 gagccgtagg aaccggccgg gataccagtg aggatggaac tatacctatt agcactgcca 240 tcttcgagaa caaggctaag acatacatga aacaaacggt gaccgtgaga agccatttat 300 aagaggtgct ccaattgaac ctgaatatgt taaaactagt atactagcaa ctgcatgctt 360 gacgtacgga ttgtccgggt catccttgcc gttccaggta tggatctcaa ggtcctccct 420 gtcacgcaga tcattgtggc tgactacccc gacatggggg tcccggcgaa agaacgaccg 480 caccctgcaa cgtcacttag tggctgtctg acagggattg attggcaatg attggcagac 540 taaccgtccc agaaacgaat catcgaactc attggtgtgc actgtgctag cgcggcgcag 600 cgacgggcgt ggctcctcgg caggggtgga catgactttc tctgtgtaga ttccttgcaa 660 ggtatctgct tgacagatgc agagatgatg ttgagtaaaa tagggtgcag agaccctgca 720 gaacctgcat cggccggcgg cgacatgtcg tcatgaccga cccgggccga gcggaattaa 780 tgaaactcgg aacagccagg ggaatccgac gctttgagtg gccaatccat gtctatccac 840 atctgcattg aagtggatga aaggtggagg agggctggtt gccaaattct gttagggctt 900 ggaatgtaag caaccggttt cggtcatgac atcatcacag cgatgacttc atcactctcg 960 gacgagcata tatagttgtg cctgtcgtcg tatctcaaca aacatcaaca acaacaacaa 1020 tcatctactg catttaccaa actcatctct acctcaatca acttaattaa tgaagt 1076 <210> SEQ ID NO 36 <211> LENGTH: 1057 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(33) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1043) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1022)..(1057) <400> SEQUENCE: 36 acttcactcg agttctcaca agcaaatccg agaaacgcaa ttgaccatat gtccttagta 60 tgagggctag caggaatact tgtttgtcga tgcataccga ctaataaaaa tggttcttct 120 ttttcgcgca gggatttggt ttttcatttt tcatttttca tattttatgt tattttattt 180 tatttttttg ttttatttta ttttattttt tttttttaaa taagactggg tgttttcgtc 240 tcccacccct gctgaggaac tgtttgatat acagaagcct gagatacttg agattatgtg 300 acaaagaaat tgtaaacctg aatgtgacta aggcagaaga gagcaggtga ggccacacaa 360 tactgtacac ccacttccga tcgtcatcga aatgagcgtt gtgggtactg gaacgacaaa 420 ccatagatcg gatctgcatc cgaataggct ttactttgta cattggtagg agtttgaatg 480 tttgaagatc cggcagatga tcacctgctg cagggaataa tttgaaacct gtccgtctgc 540 tttgacccaa ttgacgaggt acgtgatttg atgctaccca agcagtatga atgcgacgga 600

ggtggctatc cttaccttgt catatgcaac tcataacggg tagtccccat ggagccacgg 660 ctatttattt ctaagggcca acagtgaaag attgtgagat cgtatacctc ctgggtatga 720 cgcaaccagg gtgagcactg gcattgctcc aatccatatg gtggaggtat gtctccgaga 780 ccatgacgca tgtagaccct gacctgtccc tgatccaggg atctggtttg tcacttggcg 840 tattttaatt aactgcgggt cagggtcttg ccgtccagac ccaatggatg aattgcctgc 900 gtggccctgc agttccagcg atcggcacgg cacggctatg acgtcggtgg gtgttccgtt 960 tgatatttaa ccatagaaga cagccttgtt atgctggttc ctgccgcaaa ataaaggtct 1020 atccttacca cgttaagtgt caaattaatt aagaagt 1057 <210> SEQ ID NO 37 <211> LENGTH: 1061 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(33) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1047) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1023)..(1061) <400> SEQUENCE: 37 atcctactcg agtggtgcct gtgaacgagg tcaccactga taaattgtct ggatccgtaa 60 cgtctgtatc agtggatggt actgaaaagt tccaaagatt gagggcagca tatgtagcac 120 atggtggtag ctctaagtgg ttcgaaactt cgatgaatgg gcgtgatata caggggggta 180 gcacctgtat agttgatgtc agcgaaggca cgggccatgc agtgatagac aagaaaagag 240 ggattgaata aaagactcga ttctgaaggg gagtaataca tctttcggct tctcaccacc 300 ccagatgtag gcgtgtgtaa ggtaagcaag cacaacatat gctctccgcc actcaggttc 360 ccccttcaac ttcttcgtag agagtatacc caggtcatcg acagacctcc tgatagtacc 420 agactgaatc aggccaggca agtcctgagc gatttcctcc cacggggaat agtaagggtc 480 ttccaatctc cgtacgggag gaacttccgg gaggaacccg ttctggaggg agacaccgta 540 cgcaacaagt gaaatgtctt gtgtataaag catgataata tatgcacttc agtagacaac 600 ttgatattca agcctccttg aaatgatcct catgcaggta agagtagatc caagtgttta 660 tattgtattc ccaaggaggg acggaagaaa accggatcca gagggctcga gatagggcca 720 cctgcatgag caaagacggg tcatttcccg tttcacaacg accattctca ggcagaaagg 780 cagtggatta gcggaaaact tagcaaccat ccggatttct ggagtggatg ctttgttatc 840 tcacttccgg caacacggat ggtgaggcgg aagcggaacg gtatccggat ccggagggag 900 ggacaaaccg aagtgccttg tgtcacacgc cggtccatat ataaatgtta attggtgacg 960 gtcagcggat tgtttctggt gttcatccat tttttcttga tatcctggac ccagttattg 1020 cactttaatt actatccaaa atccacatta attaatgaag t 1061 <210> SEQ ID NO 38 <211> LENGTH: 1097 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(34) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1083) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1060)..(1097) <400> SEQUENCE: 38 acttcactcg aggtccacac tctaattggg atgagcattg cctttcagga acttgacttt 60 ttgtggttgc atcttttgtt gccttaccca cgcaggcttc gtttatgaga tagcgatttc 120 aggcctcaaa ttgctgtcat cacttccggc cccaaactcg tgggcggtgc ccgcccatcg 180 ctggcaagaa gcacagacta acgccttact tcgcctagtt gcacacgatg catcaagact 240 tcctcctatt tcacaacgac cagctccttg ccatttgttg tttcgaatgc ccccttcccc 300 gtgccttgca tttctaattc ccatctatac ccttttgcgc tctcacgccc gcctgactat 360 caatttttct gccagcataa ccctgcgtgc agagcggttg acagcaggct tgctaagagt 420 cttcgtctag aagaactgtc tgggtggatt ccttttcgga ttaacttttg ctattttaaa 480 agctgattgc actacggagt acacccgcaa tctgattctc actgatactc caattaaagg 540 ccactaattg ctgacatttg taacatattt gcgtttgctt tccggttgaa attggcggct 600 ttgtgctttc gatcgctagc agtctctgtg catgtacaac tgatcggcta ttcagtccta 660 ttcggaagcg gcactgagag gcggagacaa gccacagata cgacgagtct gattttgaga 720 cgggcgcatc caacgaaagc ctgcgattgc aattgaagat ctcaattggg ggactgacaa 780 cctaatacct aaacatatcg aggattaggc gccaacacga gcatgggaag ctttggtgca 840 tgcgacggct gatccatttc taacaaccgt tttgcgcgcg cgtccgaggt aaggctcccg 900 atcccttggc gctttgcatg tttcactccg atgcatctac tgtttatatg agtgcggtgg 960 gcaagctgtg cccaccgact gagcattctc aaccggccca atgctgtggg atcgatcctc 1020 ccctccaaag ttgtcgctcg gttacacttg atcggagctt gttctgttat acaccaaacc 1080 atcttaatta ataggat 1097 <210> SEQ ID NO 39 <211> LENGTH: 1142 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR product <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(35) <220> FEATURE: <221> NAME/KEY: PROMOTER <222> LOCATION: (13)..(1128) <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1118)..(1142) <400> SEQUENCE: 39 atcctactcg aggcctttgg agaatgtaat atcccttgat caccccagac tcacatccag 60 attctgactg acctcgtgca tagccatatt taacttggtc cctgatgatc aaaacagtac 120 atggaactgg ggccgaacat ttccctagcc aaaggagcat cagggattca acgtgatcag 180 tacagccttg tagctcgctg aaactgccct ggagccaagc caaagcctag acagcaaggt 240 gcatgggcga gctttgcgag gatttagttg ttatttagta tgccaagatt aagccacgca 300 gaatggctta tggccatagt agcaacaacg gccacggaac gttcttccat gtggtatggg 360 gaaagccgcc ggatatttgg ggagggcgtg attgttccgg tggctatctt tccggtggag 420 ataacggcgg tggaccatca aaactcctca gcccggattg ctcggactcc gcatcgtcaa 480 gtatcggtttc atgacagcc attcgactac ttagaagaca caaatagcta tccaacttac 540 ccggacctttg ataataatt ttcagcagat ctgtcgatct cagagacaat aatgccaccg 600 ttctgtgtgga aggcactgc gccgtggttt aacaataata aaactccaaa ttgcccaaaa 660 gctagagcgaa ggaaagtcg agaagcccag tctagactgg cgggctgatc gtccggttca 720 caacctgattg acaaatcat ctgtgaccct cgatgttccg cggcgttgag cgggtgagaa 780 tgtggatccta gtggggaag acccaattgc cgccggaaaa ggtgagcttg tgctgcacat 840 gtccggtggct caattcagg gccatgccag gacattgctc ttcaaaggga tgcgattgct 900 attccctattt acttcttta acccgctgac gggcctgtct ggaccgagaa ttcttggtca 960 agtttcagtgg agcgatcca ggaaagaaca tccgatatgg acagttgccc tcgatctcag 1020 gtctgtagtcg tgtgacaca gaggcctcta ctcgcttgcc ttgcatgtcg gagctcaaag 1080 gtgcgatgtga attaacacg aggagcgact gtctgtcgca gatgcaactt aattaatagg 1140 at 1142 <210> SEQ ID NO 40 <211> LENGTH: 39 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 40 acttcattaa ttaagttgat tgaggtagag atgagtttg 39 <210> SEQ ID NO 41 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 41 acttcactcg agataatgta ggcgacaaag tagcc 35 <210> SEQ ID NO 42 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 42 acttcattaa ttaatttgac acttaacgtg gtaagg 36 <210> SEQ ID NO 43 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 43 acttcactcg agttctcaca agcaaatccg aga 33 <210> SEQ ID NO 44 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 44 atcctactcg agtggtgcct gtgaacgagg tca 33 <210> SEQ ID NO 45 <211> LENGTH: 39

<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 45 acttcattaa ttaatgtgga ttttggatag taattaaag 39 <210> SEQ ID NO 46 <211> LENGTH: 38 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 46 atcctattaa ttaagatggt ttggtgtata acagaaca 38 <210> SEQ ID NO 47 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 47 acttcactcg aggtccacac tctaattggg atga 34 <210> SEQ ID NO 48 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 48 atcctattaa ttaagttgca tctgcgacag acagt 35 <210> SEQ ID NO 49 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 49 acttcactcg aggcctttgg agaatgtaat atccc 35 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 50 caacacctac ggcgtcaagg 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 51 taacccgggg agatgctgtt 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 52 cgatccggaa attcctcctg 20 <210> SEQ ID NO 53 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 53 gccgccgact cagcagtat 19 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 54 gtgcatgaac aacacggaga 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 55 actcagttgt gcgcctctcg 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 56 gtcgaactcg gcgtttctcc 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 57 ccggtgagga ggtgcttttc 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 58 agcatctcgc catccatcag 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 59 gccttcggac catggtatcc 20 <210> SEQ ID NO 60 <400> SEQUENCE: 60 000 <210> SEQ ID NO 61 <211> LENGTH: 1126 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: fusion gene <220> FEATURE: <221> NAME/KEY: PRIMER_BIND <222> LOCATION: (1)..(22) <220> FEATURE: <221> NAME/KEY: gene <222> LOCATION: (23)..(739) <223> OTHER INFORMATION: GFP <220> FEATURE: <221> NAME/KEY: gene <222> LOCATION: (740)..(1212) <223> OTHER INFORMATION: BLE <400> SEQUENCE: 61 gtcctgttaa ttaaccttca ccatggtgag caagggcgag gagctgttca ccggggtggt 60 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga 120 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa 180 gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag 240 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta 300 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt 360 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga 420 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat 480 catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga 540 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc 600 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa 660 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg 720 catggacgag ctgtacaagg ccaagttgac cagtgccgtt ccggtgctca ccgcgcgcga 780 cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg acttcgtgga 840 ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg cggtccagga 900 ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg acgagctgta 960 cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc cggccatgac 1020 cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg ccggcaactg 1080 cgtgcacttc gtggccgagg agcaggacta aaggcgcgcc tgaagt 1126 <210> SEQ ID NO 62 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer

<400> SEQUENCE: 62 gtcctgttaa ttaaccttca ccatggtgag caagggc 37 <210> SEQ ID NO 63 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 63 gcactggtca acttggcctt gtacagctcg tccatg 36 <210> SEQ ID NO 64 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 64 catggacgag ctgtacaagg ccaagttgac cagtgc 36 <210> SEQ ID NO 65 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 65 acttcaggcg cgcctttagt cctgctcctc ggcca 35

Claims

1. A promoter DNA sequence selected from the group consisting of: (a) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24; (b) a DNA sequence capable of hybridizing with a DNA sequence of (a); (c) a DNA sequence being at least 50% homologous to a DNA sequence of (a); (d) a variant of any of the DNA sequences of (a) to (c); and (e) a subsequence of any of the DNA sequences of (a) to (d).

2. A DNA construct comprising a promoter DNA sequence according to claim 1 operatively associated with a reporter gene conferring a selectable trait, preferably a selection marker gene.

3. An expression vector comprising a DNA construct according to claim 2, wherein the DNA construct comprises a DNA fragment, which is homologous to a DNA sequence in a predetermined target locus in the genome of a host cell, preferably the predetermined target locus comprises a highly expressed gene.

4. An expression vector comprising a DNA construct according to claim 2, wherein the DNA construct is capable of autonomous maintenance in a host cell, preferably the DNA construct comprises an AMA1-sequence.

5. A host cell comprising the DNA construct according to claim 2, or comprising a expression vector comprising the DNA construct.

6. The host cell according to claim 5, further comprising a DNA construct comprising a DNA sequence comprising a coding sequence originating from a DNA library from an organism suspected of being capable of producing one or more proteins of interest.

7. The host cell according to claim 5, wherein the host cell is a prokaryote or an eukaryote.

8. The host cell according to claim 7, wherein the host cell is selected from the following list: a Bacillus, a yeast or a filamentous fungus, preferably an Aspergillus, Penicillium or Trichoderma species.

9. The host cell according to claim 8, wherein the Aspergillus is an Aspergillus niger or Aspergillus sojae or Aspergillus oryzae species.

10. A method for isolating a DNA sequence comprising a nucleotide sequence coding for a protein of interest in a host cell, said method comprising: (a) preparing a first DNA construct comprising a promoter DNA sequence operatively associated with a reporter gene conferring a selectable trait; said promoter DNA sequence being induced when the DNA construct is present in the host cell and when a protein of interest is produced by the host cell; (b) preparing a second DNA construct comprising a DNA sequence comprising a nucleotide sequence coding for a protein of interest originating from a DNA library from an organism suspected of being capable of producing one or more proteins of interest; (c) transforming a host cell with both DNA constructs prepared in (a) and in (b); (d) culturing all the transformed host cells obtained in (c) under conditions conducive to the production of the proteins of interest as present in the DNA library; and (e) screening for transformed host cells producing a protein of interest by analysis of the proteins produced in (d).

11. The method according to claim 10, wherein the host cell is first transformed with the DNA construct prepared in step (a) and consecutively is transformed with the DNA construct prepared in step (b).

12. The method according to claim 10 wherein the promoter DNA sequence used in step (a) is selected from the group consisting of: (i) SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, or SEQ ID NO:24; (ii) a DNA sequence capable of hybridizing with a DNA sequence of (i); (iii) a DNA sequence being at least 50% homologous to a DNA sequence of (i); (iv) a variant of any of the DNA sequences of (i) to (iii); and (v) a subsequence of any of the DNA sequences of (i) to (iv).

13. The method according to claim 10, wherein the organism suspected of being capable of producing a protein of interest is a eukaryote or a prokaryote.

14. The method according to claim 10, wherein the protein of interest is an enzyme.

15. The method according to claim 10, wherein the protein of interest is a secreted protein.